Note: Descriptions are shown in the official language in which they were submitted.
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Toll-like receptors
The present invention relates to hybrid toll-like receptors, to cells
comprising such toll-like
receptors, to methods for the detection of immunostimulatory
oligodeoxynucleotides wherein
such methods use such toll-like receptors, to immunostimulatory
oligodeoxynucleotides detected
by use of this method, to the use of such immunostimulatory
oligodeoxynucleotides in medicine
and to vaccines comprising such immunostimulatory oligodeoxynucleotides.
During the past two decades, it has emerged in immunological science that the
vertebrate
immune system possesses mechanisms to detect microbial infection and to
trigger rapid immune
activation via the receptor-mediated recognition of unique and conserved
characteristics of
pathogens, the so-called pathogen-associated molecular patterns (PAMPs)
interacting with
cognate host pathogen recognition receptors (PRRs) (Iwasaki A, Medzhitov R.
2001 and
Medzhitov R., 2009).
It is now clear that certain forms of pathogen deoxyribonucleic acid (DNA) are
amongst
these PAMPs. In 1995 it was reported that non-methylated CpG motifs in
bacterial DNA trigger
murine B-cell activation (Krieg et al. 1995). This study generated for the
first time a link between
the specific recognition of bacterial immunostimulatory non-methylated CpG-
containing DNA
and the previously recognized CpG suppression as well as the widespread CpG
methylation in
mammalian DNA. The most effective B cell stimulatory non-methylated CpG
oligodeoxynucleotide (CpG ODN) was shown to possess the sequence element
GACGTT.
The next landmark paper in the field was published by Shizuo Akira's
laboratory in
Osaka/Japan (Hemmi et al. 2000). By a gene cloning and a targeted gene
knockout approach in
mice it could be unequivocally shown, that the cellular response in mice to
CpG-ODNs is
mediated by the toll-like receptor 9 (TLR9). Subsequently it was shown that
the CpG-ODNs are
agonists for TLR9 signaling predominantly via the NF kappa-B pathway
(Medzhitov 2001). In
the following decade, quite a number of studies have been published on basic
research topics and
on general potential immunotherapeutic applications (e. g. reviewed in Krieg
2002, 2003, 2006;
Klinman 2004, Vollmer 2005, Wilson et al. 2006, Kindrachuk et al. 2008, Dorn
and
Kippenberger 2008, Vollmer and Krieg 2009, Wilson et al. 2009). A number of
review articles
focus on anti-infective applications of CpG-ODNs (Krieg 2007), the use of TLR9
agonists in the
treatment of cancer (Krieg 2007, Weiner 2009), TLR9 activation for asthma and
allergy
treatment (Kline 2007, Kline and Krieg 2008, Fonseca and Kline 2009) and as
vaccine adjuvants
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( Klinman et al. 2004, Klinman 2006, Daubenberger 2007, Wagner 2009, Mutwiri
et al. 2009,
Klinman et al. 2009).
CpG ODNs have also been described and discussed as immunostimulatory agents
and vaccine
adjuvants in veterinary applications, particularly in bovines, pigs, sheep,
dogs, chicken and fish
(Babiuk et al. 2003, Carrington and Secombes 2006, Griebel et al. 2005,
Mutwiri et al. 2003,
Singh and O'Hagan 2003, Werling and Jungi 2003).
The design of specific CpG ODN's as immune modulators for both human and non-
human
species has so far been quite random. The reason for this is multi-factorial;
first of all there is no
knowledge about correlation between immune modulatory CpG motifs for human
TLR's and for
TLR's in non-human mammalian species. Secondly, there are no in vitro cell-
systems comprising
a mammalian TLR available, that have a sufficiently low signal to noise level
to allow for
selective testing of the effects of very low concentrations of CpG ODN's.
Moreover, there are no
high-throughput screening methods available and even if there were, there is
no clear correlation
between in vivo versus in vitro efficacy of CpG ODN's as immuno-modulators.
In the PCT Patent Application with Application number PCT/EP2011/074211,
unpublished at the
filing date of the present invention, the inventors have described an in vitro
cell-system that is
suitable for reproducible in vitro testing and selection of CpG ODN's as
immune modulators for
use in poultry. This system is based upon the cloning and heterologous
expression of TLR21, the
functional homologue of TLR9 in chickens.
In order to develop a comparable system for testing CpG ODN's as immune
modulators for
mammals, both for human species and for non-human species such as canine,
bovine and porcine
species, it was decided to use a comparable approach, however now based upon
the cloning and
expression of i.a. canine, bovine and porcine toll-like receptor TLR9.
To this end, a clonal line of HEK293 cells was used that contains in its
genome an
integrated version of pNifTy2-SEAP (Invivogen), that provides an NF-KB
activation reporter
gene assay based on the measurement of secreted alkaline phosphatase (SEAP)
production (See
Examples section). Such cell lines were shown by the inventors to have broad
utility in
experiments aiming at stable functional expression of TLR1/2, TLR2/6, TLR3,
TLR4, TLR5,
TLR7 and TLR8 isolated from various vertebrate (bovine, canine, porcine,
chicken) species.
Against this background it was to be expected that expression of bovine,
canine and
porcine TLR9 should be straightforward, an expectation boosted further by the
exceptionally
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well-functioning HEK293-pNifTy2-SEAP-based functional expression of the
chicken TLR9
functional homologue, TLR21.
However, repeated transfection experiments with bovine, canine and porcine
TLR9 failed to
yield cell lines that could be stimulated with known standard TLR9 agonists,
such as 2006-PTO
(TCGTCGTTTTGTCGTTTTGTCGTT) or 2007-PTO (TCGTCGTTGTCGTTTTGTCGTT),
despite the fact that the selection for plasmid introduction (G418 or
hygromycin) worked out
successfully. Only in a few experiments, weak signals were initially seen in
polyclonal
transfectant pools. These signals disappeared upon further cell line
cultivation and could not be
rescued by single cell cloning. Similar experiments were performed with
another NF-KB
1 0 activation reporter gene containing cell type, the bovine macrophage
cell line BOMAC-pNifTy2-
SEAP. The outcome was essentially the same as seen previously in HEK293-
pNifTy2-SEAP.
The reasons for the unexpected failure of functional expression (as shown by
SEAP
signal) remained unknown. An attempt to overcome the problem by using
293XL/null cells
(InvivoGen), expressing the human anti-apoptotic Bcl-XL gene did also not
result in sufficiently
1 5 high SEAP expression levels.
Thus, there still is a need for selective and sensitive systems for the
selection of CpG ODN's
having a high immuno-modulatory effect and therefore being effective in low
doses in mammals.
20 It is one of the objectives of the present invention to provide such
selective and sensitive CpG
ODN selection systems.
It was surprisingly found now that a hybrid toll-like receptor, comprising a
Toll-interleukin I
receptor-resistance (TIR) domain of poultry TLR21 and a extracellular ligand-
binding domain of
25 mammalian TLR9 is capable of overcoming the problem of low functional
expression or non-
expression as identified above.
TLRs are well-conserved type I transmembrane (TM) proteins composed of an N-
terminal signal
peptide, an extracellular ligand-binding domain containing leucine rich
repeats, a single TM
domain, and a cytoplasmic region largely comprised of the Toll-interleukin I
receptor-resistance
30 (T1R) domain.
Merely as an example: the extracellular domain of mouseTLR9 spans the region
from a.a. 1 to
820, the transmembrane domain spans the region from a.a. 820-838 and the
cytoplasmic domain
spans the region from a.a. 838 to 1032. The T1R domain spans the region from
a.a. 872-1032.
(Kajita et al, BBRC 343: 578-584 (2006)).
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The compartmentalization of TLR9 and of the poultry homologue TLR21 differs to
a certain
extent from that of TLR1, 2, 4, 5 and 6 in the sense that the part of TLR9 and
21 referred to as
the "extracellular domain" is located in the endolysosome. As a consequence
the TM region of
TLR9/21 spans the endolysosomal membrane, not the cell's plasma membrane. This
aspect and
the cell biology of TLR's in general is reviewed by Barton G.M. and Kagan,
J.C. in Nature
Reviews 9; 535-542 (2009).
Merely as examples of mammalian TLR's, the sequences of bovine, porcine and
canine TLR9
are given in SEQ ID NO's 1, 3 and 5 (nucleic acid sequence) and SEQ ID NO's 2,
4 and 6
(amino acid sequence) respectively.
It turns out that hybrid TLR's according to the invention, combining the CpG
ODN specificity of
the extracellular ligand-binding domain of mammalian TLR9 and the signaling
properties of
Toll-interleukin I receptor-resistance (TIR) domain of poultry TLR21 are
accepted by the
transfected cell without unacceptable adverse effects and at the same time
they are very well
suitable for the specific detection of CpG ODN's that are specifically immune
stimulatory to
mammalian species.
Transfection of e.g. HEK293 cells or MDCK cells with plasmids comprising DNA
encoding such
hybrid TLR's resulted in stable expression of hybrid TLR's which in turn led
to marked NF-KB
activation upon stimulation with exogenous CpG ODN's known to be active in
mammals, such
as 2006-0DN and 2007-0DN.
Thus, a first embodiment of the present invention relates to a hybrid toll-
like receptor,
characterised in that said hybrid toll-like receptor comprises a Toll-
interleukin I receptor-
resistance (T1R) domain of poultry TLR21 and a extracellular ligand-binding
domain of
mammalian TLR9.
The origin of the transmembrane region (TM region) and the non-TIR related
part of the
cytoplasmic domain is not critical, in the sense that these may independently
originate from TLR
9 or TLR21.
In a preferred form of this embodiment, the extracellular ligand-binding
domain of mammalian
TLR9 is of human, bovine, porcine or canine origin.
Examples of hybrid TLR's according to the invention where the extracellular
ligand-binding
domain of mammalian TLR9 is of bovine, porcine or canine origin, are given in
SEQ ID NO's 8,
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10 and 12 (nucleic acid sequence) and SEQ ID NO's 9, 11 and 13 (amino acid
sequence)
respectively.
An "immunostimulatory non-methylated oligodeoxynucleotide" refers to an
5 oligodeoxynucleotide, which contains a non-methylated cytidine-phosphate-
guanosine di-
nucleotide sequence that stimulates the initiation of signaling cascades
leading to activation of
transcription factors such as NF-KB or Interferon Regulatory Factor 3 (IRF3).
It is this activation
that in turn results in the expression of inflammatory cytokines and other
cellular activation
events. NF-KB binding sites and gene expression influenced by NF-KB are i.a.
described by
Schindler and Baichwal (1994).
The term oligodeoxynucleotide means a short nucleic acid polymer of
deoxynucleotides; i.e. a
molecule comprising a multitude of deoxyriboses, linked to a phosphate group
and to an
exchangeable organic base. Such an organic base is a substituted pyrimidine or
a substituted
purine. Examples are cytosine and thymine respectively adenine and guanine.
The oligonucleotides according to the invention may comprise modifications.
Examples of such
modifications are e.g. modifications in the phosphodiester internucleoside
bridge located at the 3'
and/or 5' end of a nucleoside. Such modifications relate i.a. to the
replacement of a
phosphodiester by e.g. a phosphorothioate or a phosphorodithioate.
Basically, depending upon the way of synthesis, usual common types of bonds
between two
nucleotides are: phosphodiester (PDE) bonds and phosphorothioate (PTO) bonds.
In order to
improve the stability and the immunostimulatory effect of CpG ODN's, the
building blocks of
synthetic oligodeoxynucleotides may be provided with phosphorothioates, so
that they form PTO
bonds.
Other modifications are e.g. replacements of a phosphodiester bridge by a
dephospho bridge.
Examples of dephospho bridges are methylhydroxylamine, formacetal and
dimethylenesulfone
groups.
Still other modifications are modifications that concern the replacement of a
natural nucleoside
base by a non-natural nucleoside base such as 5-fluorocytosine, 7-deaza-7-
substituted guanine, 7-
deaza-8-substituted guanine , 2-thiouracil, dihydrouracil, 5-bromo-cytosine, 6-
substituted
cytosines or N4-substituted cytosines.
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Again other modifications are modifications concerning the replacement of a
sugar unit; a B-
ribose sugar or a B-D-2'-ribose sugar unit by a modified sugar unit such as
e.g. an L-2'-
deoxyribose or 2'-L-arabinose.
A text book giving further insight in oligonucleotides is e.g. "PCR Primer: A
Laboratory
Manual", Second Edition, 2003, Edited By Carl W. Dieffenbach, National
Institute of Allergy
and Infectious Diseases; Gabriela S. Dreksler, Uniformed Services University
of the Health
Sciences, Cold Spring Harbor Laboratory Press ISBN 978-087969654-2.
For the detection of new CpG ODN' s, a system is required that comprises cells
that comprise a
hybrid TLR according to the invention
Thus, a second embodiment of the present invention relates to cells that
comprise a hybrid TLR
according to the invention.
1 5 As mentioned (vide supra), CpG ODN agonists for TLR9 signal
predominantly via the NF kappa-
B (NF-KB) pathway (Medzhitov 2001).
The detection of the effect of a CpG ODN on the occurrence and the amount of a
compound of
the (NF-KB) pathway in a cell is therefore indicative for its activity as a
PAMP.
Brownlie at al. (2009) describe an NF-KB luciferase based reporter system.
Other reporter
systems are e.g. based upon IL-8 transcript measurement or cytokine secretion
or the detection of
NO secretion.
Thus, a preferred form of this embodiment relates to a cell according to the
invention,
characterised in that said cell comprises a plasmid comprising an NF-KB
reporter gene.
Such reporter systems as mentioned above, although useful, have the
disadvantage that they are
not very sensitive. For a precise determination of the activity of existing
and newly developed
CpG ODN's, a sensitive detection system is a prerequisite.
The inventors now used a detection system in the present invention, that
turned out to be
surprisingly sensitive. This system is based upon the use of an enzyme called
secreted alkaline
phosphatase (SEAP) as a reporter enzyme encoded by the reporter gene. SEAP is
a reporter
enzyme in mammalian systems (Yang et al., 1997). In this system, SEAP
expression is controlled
by 5 NF-KB transcription factor binding sites combined with the ELAM promoter
(J. Biol. Chem.
1991, Feb 5; 266(4): 2466-73).
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Therefore, a more preferred form of this second embodiment relates to cells
according to the
invention wherein the reporter gene encodes a secreted alkaline phosphatase
(SEAP).
The SEAP system is used with para-nitrophenylphosphate (pNPP) as a substrate.
Another important improvement over existing systems is the introduction and
stable maintenance
in cells of the plasmid carrying the reporter gene.
Up till now, all detection systems used transient transfection of cells with
the reporter gene. Such
transient systems do not allow for a reliable side-by-side comparison of the
efficacy of CpG
ODN's.
Usually, stable maintenance of a plasmid is obtained by growing the cells
under the pressure of
one or more selective agents, such as antibiotics for which a resistance gene
is present on the
plasmid. Loss of the plasmid would then cause the cell that lost the plasmid
to die. Remaining
viable cells would still harbour the plasmid. Stable means that the plasmid
remains present in the
cell after several cell division cycles, preferably integrated in the cell
genome.
It is due to the introduction and stable maintenance in cells of the reporter
gene that now for the
first time a reproducible dose/response curve for CpG ODN's can be made. Such
curves are
essential if a reliable comparison between various CpG ODN's activity is to be
made.
Thus, another preferred form of this second embodiment relates to a cell
according to the
invention that comprises a plasmid encoding an NF-KB reporter gene, which
plasmid is stably
maintained in the cell. Such cells are very suitable for use in the screening
of CpG molecules,
more specifically the screening of CpG molecules according to the invention.
The Examples give ample guidance about how to obtain such a cell comprising a
plasmid
encoding a reporter gene that can be stably maintained in the cell.
Basically, any cell or cell line carrying a hybrid TLR according to the
invention that allows
introduction and preferably the stable maintenance of a plasmid carrying a NF-
KB reporter gene,
preferably the SEAP gene as described above is suitable for testing TLR9-
specific CpG ODN's.
An example of such a suitable cell line for testing TLR9-specific CpG ODN's is
the cell line
HEK293 ((ATCC number CRL-1573).
A preferred cell line for testing TLR9-specific CpG ODN's is the cell line
Madin Darby canine
kidney (ATCC number CCL-34) (MDCK).
Therefore, another preferred form of this second embodiment relates to a cell
according to the
invention wherein the cell is a HEK293 cell, preferably an MDCK cell.
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The methods and cell lines described in detail in the Examples section of the
present invention
allow for the first time to make a reliable side-by-side comparison between
various CpG ODN's
for use in mammalian species.
Thus, still another embodiment of the present invention relates to a method
for the detection of
immunostimulatory oligodeoxynucleotides according to the invention wherein
that method
comprises the steps of a) contacting an oligodeoxynucleotide with a cell
according to the
invention, b) detecting the level of product of the reporter gene.
1 0 In a preferred form of this method, the product of the reporter gene is
SEAP
As shown in the Examples below, the hybrid toll-like receptors according to
the invention have
been used extensively for the identification of new CpG ODN's.
The CpG oligodeoxynucleotides according to the invention are in most cases
active in double
1 5 digit or even sometimes in single digit nanomolar concentrations, both
in the in vitro test system
and in vivo.
The half-maximal effective concentration (EC50) of an oligodeoxynucleotide is
the amount of
oligodeoxynucleotide that is necessary to induce an amount of the reporter
enzyme SEAP (that
produces the colored product absorbing at 405 nm) in the reporter cells that
gives a half-maximal
20 absorption change over time.
The Vmax indication given is an indication of the speed with which the
chromogenic substrate of
SEAP is turned into a colored component with an absorption of 405 nm. A high
Vmax indicates
that the CpG ODN is capable of rapidly inducing a TLR-reaction.
The following new immunostimulatory non-methylated oligodeoxynucleotides were
found to
25 have a low EC50 (double or even single digit nM concentrations) and thus
to be very effective
already in very low concentrations:
[gacgtt] wherein n4
[gacgatcgtc]n wherein n3
30 [tcgtcgttttcg]n wherein n3
[tcgtcgttgtcgttttgtcgtt] wherein n2
(tx[ttcgtt]ty)n wherein 1-15, x=0-5 and y=0-5
[ttcgtNi]n wherein N1 =t or c and wherein 1-15
[Nitcgtc]n wherein N1 =t or c and wherein 1-15
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[gNicgtt], wereinnzi and Ni=a or t
[tcg]x wherein i-16
[tcgN1],, wherein N1 = c or g and i-16
[N1cgt]n wherein N1 = g or c or a or t and i-16
[acga]n wherein i-16
It should be kept in mind that all of these new immunostimulatory non-
methylated
oligodeoxynucleotides are of the phosphorothioate (PTO) type.
Therefore, again another embodiment of the present invention relates to
immunostimulatory non-
methylated PTO oligodeoxynucleotides having any of the 12 general formulae
given above.
It was generally found that the activity of the oligodeoxynucleotides
increases when n increases.
This effect is leveling when n increases. Basically, the number n of the
backbone structure should
therefore be at least the number of n as indicated. Preferably, the upper
range of n is n < 100,
merely because of the fact that the longer the synthetic sequence the more
difficult it is to make.
In practice therefore a more preferable upper range of n is n < 40, even more
preferable n < 20.
It is very well possible to link an oligodeoxynucleotide according to the
invention to a carrier or
hapten, via a reactive chemical group. Such linkage enhances the
immunostimulatory effect of
the combined molecules.
Mere examples of such components are e.g. digoxigenin, aminohexyl-, Texas red
and biotin.
Preferred carriers or haptens are 3'- and 5'-labeled Texas red and 5'-labeled
digoxigenin. The
linkage of oligodeoxynucleotides to haptens/carriers is well-known in the art.
Thus, a preferred form of this embodiment relates to an immunostimulatory non-
methylated PTO
oligodeoxynucleotide having one of the 12 general formulae given above wherein
said
oligodeoxynucleotide is coupled to a carrier or hapten.
Another embodiment of the invention relates to a vector comprising an
immunostimulatory non-
methylated oligodeoxynucleotide according to the invention. Such a vector can
be a nucleic acid
molecule such as a plasmid, a virus, a bacteriophage or any other vector used
in molecular
biology. Merely as an example: a vector comprising an immunostimulatory non-
methylated
oligodeoxynucleotide can e.g. be a DNA molecule such as a plasmid that can be
multiplied in
bacteria, into which an immunostimulatory non-methylated oligodeoxynucleotide
according to
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the invention has been cloned. Such a plasmid preferably has an active origin
of replication,
causing high numbers of the plasmid to be present in the host. Growing such
bacteria on a large
scale followed by isolation of the plasmids provides an alternative for the
synthetic production of
the immunostimulatory non-methylated oligodeoxynucleotide according to the
invention. It
5 should be kept in mind that this embodiment only applies to
immunostimulatory non-methylated
oligodeoxynucleotides of the PDE type.
One of the aims of the present invention is to provide new CpG ODN's that can
be used as
successful immunostimulating components in vaccines that prevent or combat
infectious disease
10 together with an antigen component or genetic information encoding an
antigen component, and
a pharmaceutically acceptable carrier.
In general, the term antigen component refers to a composition of matter that
comprises at least
one epitope that can induce, stimulate or enhance an immune response when
administered to a
human or an animal.
The antigen component may be any kind of antigen component but preferably is
derived from a
micro-organism or virus that in its wild-type form is pathogenic to humans or
animals.
The antigen component can be the whole pathogen, preferably in an inactivated
or attenuated
form, an extract of the pathogen or (an immunogenic part of) an immunogenic
protein of the
pathogen.
If the antigen component is (an immunogenic part of) an immunogenic protein of
the pathogen,
that immunogenic protein is preferably expressed in and recovered from in
vitro cultured cells.
Therefore, another embodiment relates to a vaccine for preventing or combating
infectious
disease characterised in that said vaccine comprises an immunostimulating
amount of an
oligodeoxynucleotide according to the invention and/or a vector according to
the invention, an
immunogenic amount of an antigen component or genetic information encoding an
antigen
component, and a pharmaceutically acceptable carrier.
The skilled person will understand that the immunostimulating amount of the
oligodeoxynucleotide and the immunogenic amount of the antigen component are
strongly
interrelated. It is one of the merits of the present invention that new
oligodeoxynucleotide are
provided that can lower the amount of antigen component that is necessary to
prevent or combat
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infectious disease.
The amount of antigen component that is necessary to prevent or combat
infectious disease is
referred to as the immunogenic amount of the antigen component.
An immunostimulating amount of the oligodeoxynucleotide is the amount that is
capable of
decreasing the immunogenic amount of the antigen component, i.e. the amount of
the antigen
component that is necessary to prevent or combat an infectious disease.
So basically, the wording "immunostimulating amount of the
oligodeoxynucleotide" and
"immunogenic amount" must be seen in relation to each other.
It goes without saying that, if the vaccine comprises genetic information
encoding an antigen
component, the amount of antigen component expressed by this genetic
information should be
enough to prevent or combat infectious disease, i.e.; it must be an
immunogenic amount.
The fact that the non-methylated oligodeoxynucleotides according to the
invention are
immunostimulatory, means that they enhance the immunological efficacy of
antigen components
in vaccines. For that reason, vaccines according to the invention will in many
cases comprise less
of the antigen component or the genetic information encoding the antigen
component than would
be the case if no oligodeoxynucleotides according to the invention would be
present.
In some cases an antigen component as such, without the addition of
immunostimulatory
oligonucleotides, may have such low immunogenic properties that high amounts
must be given
anyway, albeit without reaching the desired immunogenic level. In such cases,
the antigen
component can be given in the usual high concentration, however now together
with an
oligodeoxynucleotide according to the invention in order to so obtain the
desired level of
immunogenicity.
Thus, the amount of the antigen component or the genetic information encoding
the antigen
component to be administered with an oligonucleotide according to the
invention would as a rule
of thumb be equal or below the amount given in the absence of the
oligonucleotide. The skilled
person involved in the manufacturing of a specific vaccine, would know that
amount for that
specific vaccine. Also, the Examples give e.g. guidance for the amount of
antigen components to
be used, e.g. for a rabies vaccine for canine species.
The amount of the oligodeoxynucleotide according to the invention that needs
to be administered
together with the antigen component or the genetic information encoding the
antigen component
depends both on the selected oligodeoxynucleotide and the antigen component.
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A very suitable amount of oligodeoxynucleotide according to the invention
would usually vary
between 1 and 100 nanomol. Very good in vivo results have e.g. been obtained
with 5-50 ug of
oligodeoxynucleotides according to the invention with an average length of 30
deoxynucleotides
that were shown to be active in in vitro tests in the nanomolar range.
If an oligodeoxynucleotide is chosen from the group of oligodeoxynucleotides
that are active in
the picomolar range, the skilled person would realise that amounts below,
possibly far below, 1
nanomol, i.e. picomol amounts (e.g. 100-1000 ng), would be worth testing
before testing
nanomolar amounts. The skilled person should be aware of the fact that there
may be an optimal
amount for each of the oligodeoxynucleotides according to the invention.
Vaccines according to the invention comprise a pharmaceutically acceptable
carrier. The nature
of this carrier depends i.a. upon the route of administration. If the
administration route is through
the oral or intranasal route, the carrier could be as simple as sterile water,
a physiological salt
solution or a buffer. If injection is the preferred route, the carrier should
preferably be isotonic
and have pH restrictions that make it suitable for injection. Such carriers
however are extensively
known in the art.
Vaccines according to the invention may, in addition to the antigen component
or the genetic
information encoding the antigen component, and an oligodeoxynucleotide
according to the
invention, comprise an adjuvant. Adjuvants in general are substances that
boost the immune
response of the host in a non-specific manner.
Many adjuvants are known in the art to be suitable, such as Freund's Complete
and Incomplete
adjuvant, vitamin E, non-ionic block polymers and polyamines such as dextran
sulphate,
carbopol and pyran, alum hydroxide. Also frequently used are alumin phosphate,
saponins,
vegetable oils such as tocopherol and mineral oils. Very efficient adjuvants
are oil-in-water
emulsions and especially water-in-oil emulsions, further also referred to as
oil-in-water adjuvants
and water-in-oil adjuvants. Such emulsions are well-known in the art. Thus,
preferably, the
vaccine comprises a water-in-oil adjuvant.
Preferably the antigen component is, or is derived from a virus or micro-
organism that in its wild-
type form is pathogenic to humans, porcine, canine or bovine species.
For a large number of pathogens, vaccines are commercially available. These
pathogens are listed
below.
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Thus, more preferably said virus or micro-organism is selected from the group
consisting of
human papillomavirus, a bacterium causing tuberculosis, diphtheria, pertussis,
tetanus,
pneumonia or meningitis, measles virus, poliomyelitis virus, hepatitis B
virus, Leptospira,
Mycobacterium hyopneumomiae, Bovine respiratory syncytium virus, Foot-and-
mouth disease
virus, Bovine Viral Diarrhoea virus, Porcine Respiratory and Reproductive
Syndrome virus,
canine parvovirus, canine parainfluenza virus, canine coronavirus, canine
distemper virus, canine
adenovirus, porcine Circovirus 2, Bovine Herpesvirus, rabies virus, classical
swine fever virus,
equine Herpesvirus, porcine parvovirus, Escherichia coli, Pasteurella (i.a. P.
multocida),
Bordetella (i.a. B. bronchiseptica), Pseudorabies virus, Erysipelothrix,
Haemophilus parasuis,
1 0 Bovine parainfluenza virus, Mannheimia (i.a. M. haemolytica),
Fusobacterium, Lawsonia
intracellularis, Streptococcus equi, Chlamidophila, Actinobacillus
pleuropneumoniae, Brucella
abortus, Dictyocaulis, Toxoplasma gondii, Babesia (i.a. B. canis), Neospora,
Giardia,
Sarcocystis and Leishmania.
Again another embodiment of the present invention relates to an
immunostimulatory non-
methylated oligodeoxynucleotide according to the invention in combination with
an
immunological amount of an antigen component or genetic information encoding
an antigen
component, and a pharmaceutically acceptable carrier for use as a medicament.
Still another embodiment of the present invention relates to an
immunostimulatory non-
methylated oligodeoxynucleotide according to the invention in combination with
an
immunological amount of an antigen component or genetic information encoding
an antigen
component, and a pharmaceutically acceptable carrier for use in preventing or
combating
infectious disease in mammalian species, preferably human, porcine, bovine and
canine species.
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Legend to the figures:
Figure 1: MDCK (canine) ¨ pNifTyhyg: reactivity with different PAMPs. Vertical
axis:
mOD450nm/min.
Figure 2: MDCK (canine) ¨ pNifTyhyg: reactivity with different PAMPs. Vertical
axis:
mOD450nm/min.
Figure 3: MDcanK-pNifTyhyg-single cell clones 1-46-huTNF-alpha stimulation.
Horizontal axis: from left to right; clone 1 to clone 46, pool and control
Figure 4: PCR "sewing" strategy.
Figure 5: canTLR9-21MDCKpNifTyhyg-single cell clones 1-54-2006-PTO
stimulation.
Horizontal axis: from left to right; clone 1 to clone 54, followed by "canTLR9-
TLR21-
pool" (the polyclonal cell line before single cell cloning). The reactivity is
given in pairs
of bars: the left bar (gray) is the level of stimulation with 1 microM of 2006-
PTO, the
right bar (black is the control)
Figure 6-18: MDCK-pNifTyhyg-p1RESpuro-canTLR9-21fusion: stimulation with
several
PAMPs as indicated
Figure 19: MDCK-pNifTyhyg-pigTLR9/TLR21-single cell clones 1-75 ODN-2006-PT0
stimulation. Horizontal axis: from left to right; clone 1 to clone 75, pool,
"canis-
TLR9/21-clone17" (dog TLR9-21 fusion clonal cell line (no 17) as a positive
control)
and "MDCK-pNifTyhyg" (the basal MDCK cell line used for the transfection
experiment).
Figure 20-31: MDCK-pNifTyhyg-pigTLR9/TLR21-fusion, tested with different PAMPs
as indicated.
Figure 32: Antibody titer of Nobivac rabies vaccine with and without hio-tcg-8-
PT0 as
indicated.
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Krieg A.M., 2002. CpG motifs in bacterial DNA and their immune effects. Annu.
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2006. Immune mechanisms and therapeutic potential of CpG
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Examples
Example 1.
Gene cloning of bovine toll-like receptor 9 (TLR-9)
Fresh bovine spleen was obtained from a local slaughterhouse as a source of
bovine
TLR9 messenger RNA (mRNA). Total RNA was prepared from bovine spleen tissue
essentially
as outlined by Chomczynski and Sacchi (1987) using a commercial kit and its
instructions
(TRIZOL , GIBCOBRL). From the bovine spleen total RNA, first strand cDNA was
synthesized, essentially as described by the supplier of the reverse
transcriptase (-Expand Reverse
Transcriptase, Roche). Primer were designed for the polymerase chain reaction
(PCR)
amplification of bovine TLR9 (Genbank AY859726) from the start codon region to
a 3'UTR
region downstream of the stop codon (Bov-TLR9-for and Bov-TLR9-rev, see
below). However,
initial PCR experiments (Expand High Fidelity PCR kit, Roche) using bovine
spleen first strand
cDNA aiming at the amplification of the full length product (expected: ¨ 3100
bp) proved to be
repeatedly negative. Closer inspection of the bovine TLR9 gene indicated a
high GC content (-
64%). Therefore, it was decided to test a PCR system optimized for this
particular problem
(Advantagem4 GC2, Clontech) . The corresponding PCR reaction yielded a weak
DNA fragment
of the expected size (¨ 3100 bp).
Primer sequences:
Bov-TLR9-for GGGTACCATGGGCCCCTACTGTGCCCCGCAC
Bov-TLR9-rev GTCTAGAGTCTGTGCTATTCGGCTGTCGTGG
Cloning of the PCR fragment into pCR2.1-Topo (Invitrogen) was performed, and
four
clones were sequenced to identify a PCR error-free version (pCR2.1-Topo-
bovineTLR9). By
exploiting primer-introduced KpnI and XbaI restriction enzyme sites, the
bovine TLR9 insert was
excised, agarose gel-purified and subcloned into the KpnI/XbaI-cut mammalian
expression
vectors pcDNA3.1(neo) and pcDNA3.1(hyg) (both Invitrogen), yielding
pcDNA3.1(neo)-
bovineTLR9 and pcDNA3.1(hyg)-bovineTLR9, respectively. The corresponding
inserts were
resequenced (see below).
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pcDNA3.1 insert sequence bovine TLR9 (primer sequences underlined, start/stop
codons
highlighted bold), 3090 bp.
GGTACCATGGGCCCCTACTGTGCCCCGCACCCCCTTTCTCTCCTGGTGCAGGCGGCGGCACTGGCAGCGGCC
CTGGCCGAGGGCACCCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCATGGTCAGGTGGACTGCAACTGG
CTGTTCCTGAAGTCTGTGCCGCACTTTTCGGCTGGAGCCCCCCGGGCCAATGTCACCAGCCTCTCCTTAATC
TCCAACCGCATCCACCACTTGCATGACTCTGACTTCGTCCACCTGTCCAACCTGCGGGTCCTCAACCTCAAG
TGGAACTGCCCGCCGGCCGGCCTCAGCCCCATGCACTTCCCCTGCCGTATGACCATCGAGCCCAACACCTTC
CTGGCTGTGCCCACCCTGGAGGAGCTGAACCTGAGCTACAACGGCATCACGACCGTGCCTGCCCTGCCCAGT
TCCCTCGTGTCCCTGTCGCTGAGCCACACCAGCATCCTGGTGCTAGGCCCCACCCACTTCACCGGCCTGCAC
GCCCTGCGCTTTCTGTACATGGACGGCAACTGCTACTACATGAACCCCTGCCCGCGGGCCCTGGAGGTGGCC
CCAGGCGCCCTCCTCGGCCTGGGCAACCTCACGCACCTGTCGCTCAAGTACAACAACCTCACGGAGGTGCCC
CGCCGCCTGCCCCCCAGCCTGGACACCCTGCTGCTGTCCTACAACCACATTGTCACCCTGGCACCCGAGGAC
CTGGCCAACCTGACTGCCCTGCGCGTGCTTGACGTGGGTGGGAACTGCCGCCGCTGCGACCACGCCCGCAAC
CCCTGCAGGGAGTGCCCAAAGAACTTCCCCAAGCTGCACCCTGACACCTTCAGTCACCTGAGCCGCCTCGAA
GGCCTGGTGTTGAAGGACAGTTCTCTCTACAAACTAGAGAAAGATTGGTTCCGCGGCCTGGGCAGGCTCCAA
GTGCTCGACCTGAGTGAGAACTTCCTCTATGACTACATCACCAAGACCACCATCTTCAACGACCTGACCCAG
CTGCGCAGACTCAACCTGTCCTTCAATTACCACAAGAAGGTGTCCTTCGCCCACCTGCACCTAGCGTCCTCC
TTTGGGAGTCTGGTGTCCCTGGAGAAGCTGGACATGCACGGCATCTTCTTCCGCTCCCTCACCAACATCACG
CTCCAGTCGCTGACCCGGCTGCCCAAGCTCCAGAGTCTGCATCTGCAGCTGAACTTCATCAACCAGGCCCAG
CTCAGCATCTTTGGGGCCTTCCCGAGCCTGCTCTTCGTGGACCTGTCGGACAACCGCATCAGCGGAGCCGCG
ACGCCAGCGGCCGCCCTGGGGGAGGTGGACAGCAGGGTGGAAGTCTGGCGATTGCCCAGGGGCCTCGCTCCA
GGCCCGCTGGACGCCGTCAGCTCAAAGGACTTCATGCCAAGCTGCAACCTCAACTTCACCTTGGACCTGTCA
CGGAACAACCTGGTGACAATCCAGCAAGAGATGTTTACCCGCCTCTCCCGCCTCCAGTGCCTGCGCCTGAGC
CACAACAGCATCTCGCAGGCGGTTAATGGCTCCCAGTTCGTGCCGCTGACCAGCCTGCGAGTGCTCGACCTG
TCCCACAACAAGCTGGACCTGTACCATGGGCGCTCATTCACGGAGCTGCCGCAGCTGGAGGCACTGGACCTC
AGCTACAACAGCCAGCCCTTCAGCATGCAGGGCGTGGGCCACAACCTCAGCTTCGTGGCCCAGCTGCCCTCC
CTGCGCTACCTCAGCCTTGCGCACAATGGCATCCACAGCCGCGTGTCACAGAAGCTCAGCAGCGCCTCGTTG
CGCGCCCTGGACTTCAGCGGCAACTCCCTGAGCCAGATGTGGGCCGAGGGAGACCTCTATCTCTGCTTTTTC
AAAGGCTTGAGGAACCTGGTCCAGCTGGACCTGTCCGAGAACCATCTGCACACCCTCCTGCCTCGTCACCTG
GACAACCTGCCCAAGAGCCTGCGGCAGCTGCGTCTCCGGGACAATAACCTGGCCTTCTTCAACTGGAGCAGC
CTGACCGTCCTGCCCCGGCTGGAAGCCCTGGATCTGGCAGGAAACCAGCTGAAGGCCCTGAGCAACGGCAGC
CTGCCGCCTGGCATCCGGCTCCAGAAGCTGGACGTGAGCAGCAACAGCATCGGCTTCGTGATCCCCGGCTTC
TTCGTCCGCGCGACTCGGCTGATAGAGCTTAACCTCAGCGCCAATGCCCTGAAGACAGTGGATCCCTCCTGG
TTCGGTTCCTTAGCAGGGACCCTGAAAATCCTAGACGTGAGCGCCAACCCGCTCCACTGCGCCTGCGGGGCG
GCCTTTGTGGACTTCCTGCTGGAGAGACAGGAGGCCGTGCCCGGGCTGTCCAGGCGCGTCACATGTGGCAGT
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CCGGGCCAGCTCCAGGGCCGCAGCATCTTCACACAGGACCTGCGCCTCTGCCTGGATGAGACCCTCTCCTTG
GACTGCTTTGGCCTCTCACTGCTAATGGTGGCGCTGGGCCTGGCAGTGCCCATGCTGCACCACCTCTGTGGC
TGGGACCTCTGGTACTGCTTCCACCTGTGTCTGGCCCATTTGCCCCGACGGCGGCGGCAGCGGGGCGAGGAC
ACCCTGCTCTATGATGCCTTCGTGGTCTTCGACAAGGTGCAGAGTGCAGTGGCTGATTGGGTGTACAACGAG
5 CTCCGCGTGCAGCTGGAGGAGCGCCGGGGGCGCCGGGCGCTCCGCCTCTGCCTGGAGGAGCGAGACTGGCTC
CCTGGTAAGACGCTCTTCGAGAACCTGTGGGCCTCGGTCTACAGCAGCCGCAAGACCATGTTCGTGCTGGAC
CACACGGACCGGGTCAGCGGCCTCCTGCGCGCCAGCTTCCTGCTGGCCCAGCAGCGCCTGTTGGAGGACCGC
AAGGACGTCGTAGTGCTGGTGATCCTGCGCCCCGCCGCCTATCGGTCCCGCTACGTGCGGCTGCGCCAGCGC
CTCTGCCGCCAGAGCGTCCTCCTCTGGCCCCACCAGCCCAGTGGCCAGGGTAGTTTCTGGGCCAACCTGGGC
10 ATAGCCCTGACCAGGGACAACCGTCACTTCTATAACCGGAACTTCTGCCGGGGCCCCACGACAGCCGAATAG
CACAGACTCTAGA
MGPYCAPHPLSLLVQAAALAAALAEGTL PAFL PCELQPHGQVDCNWLFLKSVPHFSAGAPRANVT SLSL I SN
RI HHLHDS DFVHL SNLRVLNLKWNC P PAGL S PMHF PCRMT IE PNT FLAVP TLEELNL S YNGI
TTVPALPS SL
15 VS L SL SHT S I LVLGP THFTGLHALRFLYMDGNCYYMNPCPRALEVAPGAL LGLGNL THL
SLKYNNLTEVPRR
L P P SL DTL LL SYNHIVTLAPEDLANLTALRVL DVGGNCRRCDHARNPCRECPKNFPKLHPDT FSHL
SRLEGL
VLKDS SLYKLEKDWFRGLGRLQVLDL SENFLYDY I TKT T I FNDLTQLRRLNL S FNYHKKVSFAHLHLAS
S FG
SLVSLEKL DMHG I FFRSL TN I T LQS LTRL PKLQSLHLQLNF INQAQL S I FGAF P SL LFVDL
S DNRI SGAATP
AAALGEVDSRVEVWRLPRGLAPGPLDAVSSKDFMP SCNLNFTLDL SRNNLVT I QQEMFTRL SRLQCLRL SHN
20 Si SQAVNGSQFVPLT SLRVLDL SHNKLDLYHGRSFTEL PQLEALDLSYNSQPFSMQGVGHNL
SFVAQLPSLR
YL SLAHNGIHSRVSQKLS SAS LRALDFSGNS L SQMWAEGDLYLCFFKGLRNLVQLDL SENHLHT LL PRHL
DN
LPKSLRQLRLRDNNLAFFNWSSLTVLPRLEALDLAGNQLKAL SNGSL P PG IRLQKL DVS SNS IGFVI
PGFFV
RATRL IELNLSANALKTVDP SWFGS LAGTLK I LDVSANPLHCACGAAFVDFLLERQEAVPGL SRRVTCGS
PG
QLQGRS I FTQDLRLCLDE TL SL DCFGL S LLMVALGLAVPMLHHLCGWDLWYCFHLCLAHL
PRRRRQRGEDTL
LYDAFVVFDKVQSAVADWVYNELRVQLEERRGRRALRLCLEERDWLPGKTLFENLWASVYSSRKTMFVLDHT
DRVSGLLRASFL LAQQRL LEDRKDVVVLVI LRPAAYRSRYVRLRQRLCRQSVL LWPHQP SGQGSFWANLG IA
LTRDNRHFYNRNFCRGPTTAE .
The translated sequence was aligned with 10 bovine TLR9 full length cDNA
sequences
deposited in Genbank (September 2011, P8-141108-prot-280109.pro Bov-TLR9-
NM_183081.pro B ov-rom-TLR9 -EF 076723 .pro Bov-ang-TLR9-EF076724.pro B ov-
braf-
TLR9-EF 076725 .pro Bov-brah-TLR9-EF076726.pro Bov-char-TLR9-EF076727.pro Bov-
hol-
TLR9-EF076728.pro Bov-lim-TLR9-EF076729.pro Bov-pied-TLR9-EF076731.pro B ov-
TLR9-
AY859726.pro). Alignment (ClustalW; DNAStar) of the 11 bovine TLR9 polypeptide
sequences
showed polymorphisms at 7 positions. In 5 cases the translated sequence of our
TLR9 clone (P8-
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141108-prot-280109) conformed to the majority polypeptide sequence. In the two
other positions,
identical residues were found as in the Genbank sequence AY859726.
Therefore, it is concluded that a correct version of bovine TLR9 has been
cloned.
Example 2
Gene cloning of porcine toll-like receptor 9 (TLR-9)
Fresh porcine spleen was obtained from a local slaughterhouse as a source of
porcine
TLR9 messenger RNA (mRNA). Total RNA was prepared from porcine spleen tissue
essentially
as outlined by Chomczynski and Sacchi (1987) using a commercial kit and its
instructions
(TRIZOL , GIBCOBRL). From the porcine spleen total RNA, first strand cDNA was
synthesized, essentially as described by the supplier of the reverse
transcriptase ( Expand Reverse
Transciptase, Roche).
Primer were designed for the polymerase chain reaction (PCR) amplification of
porcine
TLR9 (Genbank NM 213958) from the start codon region to a 3'UTR region
downstream of the
stop codon (PigTLR9forl and PigTLR9revl, see below). However, initial PCR
experiments
using porcine spleen first strand cDNA aiming at the amplification of the full
length product
(expected: 3246 bp) proved to be repeatedly negative. Therefore, it was
decided to take advantage
of a unique Xho I site bisecting the porcine TLR9 gene into two approximately
equally sized
fragments (1501 bp and 1745 bp, respectively), that were more amenable for a
PCR approach. To
this end primer were designed upstream of the Xho I site in a forward
direction and downstream
of the Xho I site in a reverse direction (PigTLR9Xh0I-for and PigTLR9Xh0I-rev,
see below), to
give rise to PCR fragments overlapping near the unique XhoI site.
Primer sequences:
PigTLR9for1 GAAGCTTACCATGGGCCCCCGCTGCACCCTGCACCCC
PigTLR9rev1 GGCGGCCGCTTACATGCCAGGCTGGGGGGTGGGGTG
PigTLR9Xhol-for GGTGACAATCCAGTCGGAGATGTTTGCTCG
PigTLR9Xh0l-rev GGTCCAGCTTGTTGTGGGACAGGTCCAGC
PCR reactions (Expand High Fidelity PCR kit, Roche) were performed with the
primer
pairs PigTLR9forl/ PigTLR9Xh0I-rev (for amplification of the 5' gene fragment)
and
PigTLR9Xh0I-for/ PigTLR9rev1 (for amplification of the 3' gene fragment). The
corresponding
PCR products were agarose gel-purified, cloned into pCR2.1-topo (Invitrogen),
and three clones
each were sequenced to identify PCR error-free versions. By exploiting vector-
based and PCR
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fragment-based XhoI sites, the corresponding 5'- and 3'-PCR fragments were
joined to the full
length porcine TLR9 gene. From the corresponding construct (pCR2.1-Topo-
porcineTLR9) the
insert was excised by HindIIUNotI digestion, agarose gel-purified and
subcloned into the
HindIII/NotI-cut mammalian expression vectors pcDNA3.1(neo) and pcDNA3.1(hyg)
(both
Invitrogen), yielding pcDNA3.1(neo)-porcineTLR9 and pcDNA3.1(hyg)-porcineTLR9,
respectively. The corresponding inserts were resequenced (see below).
pcDNA3.1 insert sequence porcine TLR9 (primer sequences underlined, start/stop
codons
highlighted bold, unique XhoI site highlighted)
GAAGCTTACCATGGGCCCCCGCTGCACCCTGCACCCCCTTTCTCTCCTGGTGCAGGTGACAGCGCTGGCTGC
GGCTCTGGCCCAGGGCAGGCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCACGGCCTGGTGAACTGCAA
CTGGCTCTTCCTGAAGTCCGTGCCCCACTTCTCGGCGGCAGCGCCCCGGGCCAACGTCACCAGCCTCTCCTT
ACTCTCCAACCGCATCCACCACCTGCACGACTCTGACTTCGTCCACCTGTCCAGCCTACGAACTCTCAACCT
CAAGTGGAACTGCCCGCCGGCTGGCCTCAGCCCCATGCACTTCCCCTGCCACATGACCATCGAGCCCAACAC
CTTCCTGGCCGTGCCCACCCTGGAGGAGCTGAACCTGAGCTACAACAGCATCACGACCGTGCCTGCCCTGCC
CGACTCCCTCGTGTCCCTGTCGCTGAGCCGCACCAACATCCTGGTGCTAGACCCCACCCACCTCACTGGCCT
ACATGCCCTGCGCTACCTGTACATGGATGGCAACTGCTACTACAAGAACCCCTGCCAGGGGGCGCTGGAGGT
GGTGCCGGGTGCCCTCCTCGGCCTGGGCAACCTCACACATCTCTCACTCAAGTACAACAATCTCACGGAGGT
GCCCCGCAGCCTGCCCCCCAGCCTGGAGACCCTGCTGTTGTCCTACAACCACATTGTCACCCTGACGCCTGA
GGACCTGGCCAATCTGACTGCCCTGCGCGTGCTTGATGTGGGGGGGAACTGCCGCCGCTGTGACCATGCCCG
CAACCCCTGCAGGGAGTGCCCAAAGGACCACCCCAAGCTGCACTCTGACACCTTCAGCCACCTGAGCCGCCT
CGAAGGCCTGGTGTTGAAAGACAGTTCTCTCTACAACCTGGACACCAGGTGGTTCCGAGGCCTGGACAGGCT
CCAAGTGCTGGACCTGAGTGAGAACTTCCTCTACGACTGCATCACCAAGACCACGGCCTTCCAGGGCCTGGC
CCGACTGCGCAGCCTCAACCTGTCCTTCAATTACCACAAGAAGGTGTCCTTTGCCCACCTGCACCTGGCACC
CTCCTTTGGGCACCTCCGGTCCCTGAAGGAGCTGGACATGCATGGCATCTTCTTCCGCTCGCTCAGTGAGAC
CACGCTCCAACCTCTGGTCCAACTGCCTATGCTCCAGACCCTGCGCCTGCAGATGAACTTCATTAACCAGGC
CCAGCTCAGCATCTTTGGGGCCTTCCCTGGCCTGCTGTACGTGGACCTATCGGACAACCGCATCAGCGGAGC
TGCAAGGCCAGTGGCCATTACTAGGGAGGTGGATGGTAGGGAGAGGGTCTGGCTGCCTTCCAGGAACCTCGC
TCCACGTCCACTGGACACTCTCCGCTCAGAGGACTTCATGCCAAACTGCAAGGCCTTCAGCTTCACCTTGGA
CCTGTCTCGGAACAACCTGGTGACAATCCAGTCGGAGATGTTTGCTCGCCTCTCACGCCTCGAGTGCCTGCG
CCTGAGCCACAACAGCATCTCCCAGGCGGTCAATGGCTCTCAGTTTGTGCCGCTGACCAGCCTGCGGGTGCT
GGACCTGTCCCACAACAAGCTGGACCTGTATCACGGGCGCTCGTTCACGGAGCTGCCGCGCCTGGAAGCACT
GGACCTCAGCTACAATAGCCAGCCCTTTACCATGCAGGGTGTGGGCCACAACCTCAGCTTCGTGGCCCAGCT
GCCCGCCCTGCGCTACCTCAGCCTGGCGCACAATGACATCCATAGCCGAGTGTCCCAGCAGCTCTGTAGCGC
CTCACTGTGCGCCCTGGACTTTAGCGGCAACGATCTGAGCCGGATGTGGGCTGAGGGAGACCTCTATCTCCG
CTTCTTCCAAGGCCTAAGAAGCCTAGTCTGGCTGGACCTGTCCCAGAACCACCTGCACACCCTCCTGCCACG
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TGCCCTGGACAACCTCCCCAAAAGCCTGAAGCATCTGCATCTCCGTGACAATAACCTGGCCTTCTTCAACTG
GAGCAGCCTGACCCTCCTGCCCAAGCTGGAAACCCTGGACTTGGCTGGAAACCAGCTGAAGGCCCTAAGCAA
TGGCAGCCTGCCATCTGGCACCCAGCTGCGGAGGCTGGACCTCAGTGGCAACAGCATCGGCTTTGTGAACCC
TGGCTTCTTTGCCCTGGCCAAGCAGTTAGAAGAGCTCAACCTCAGCGCCAATGCCCTCAAGACAGTGGAGCC
CTCCTGGTTTGGCTCGATGGTGGGCAACCTGAAAGTCCTAGACGTGAGCGCCAACCCTCTGCACTGTGCCTG
TGGGGCGACCTTCGTGGGCTTCCTGCTGGAGGTACAGGCTGCCGTGCCTGGGCTGCCCAGCCGCGTCAAGTG
TGGCAGTCCGGGGCAGCTCCAGGGCCATAGCATCTTTGCGCAAGACCTGCGCCTCTGCCTGGATGAGACCCT
CTCGTGGAACTGTTTTGGCATCTCGCTGCTGGCCATGGCCCTGGGCCTGGTTGTGCCCATGCTGCACCACCT
CTGCGGCTGGGACCTCTGGTACTGCTTCCACCTGTGCCTGGCCTGGCTGCCCCACCGAGGGCAGCGGCGGGG
CGCAGACGCCCTGTTCTATGATGCCTTCGTGGTCTTTGACAAAGCTCAGAGTGCTGTGGCCGACTGGGTGTA
CAACGAGCTGCGGGTGCAGCTGGAGGAGCGCCGTGGGCGCCGCGCACTGCGCCTGTGCCTGGAGGAGCGAGA
CTGGTTACCTGGCAAGACGCTCTTCGAGAACCTGTGGGCCTCAGTCTACAGCAGCCGCAAGACCCTGTTTGT
GC TGGCCCACACGGACCGTGTCAGCGGCCTCT TGCGTGCCAGTTTCC TGC TGGCCCAGCAGCGCC TGCTGGA
GGACCGCAAGGACGTTGTAGTGCTGGTGATCCTGCGCCCCGATGCCTACCGCTCCCGCTACGTGCGGCTGCG
CCAGCGCCTCTGCCGCCAGAGTGTCCTCCTCTGGCCCCACCAGCCCCGTGGGCAGGGCAGCTTCTGGGCCCA
GC TGGGCACAGCCCTGACCAGGGACAACCACCACT TCTATAACCGGAACT TCTGCCGGGGCCCCACGACAGC
CGAATAGCACTGAGTGACAGCCCAGTTGCCCCAGCCCCCCTGGATTTGCCTCTCTGCCTGGGGTGCCCCAAC
CTGCTTTGCTCAGCCACACCACTGCTCTGCTCCCTGTTCCC CACCCCACCCCCCAGCCTGGCATG
MGPRCTLHPLSLLVQVTALAAALAQGRL PAFL PCELQPHGLVNCNWLFLKSVPHFSAAAPRANVT SL SLL SN
RI HHLHDS DFVHL S S LRT LNLKWNC P PAGL S PMHF PCHMT IE PNT FLAVP TLEELNL S YNS
I TTVPALPDSL
VS L SL SRTNI LVLDP THL TGLHALRYLYMDGNCYYKNPCQGALEVVPGALLGLGNL THL
SLKYNNLTEVPRS
LPPSLETLLLSYNHIVTLTPEDLANLTALRVLDVGGNCRRCDHARNPCRECPKDHPKLHSDTFSHLSRLEGL
VLKDS SLYNLDTRWFRGL DRLQVLDL SENFLYDC I TKTTAFQGLARLRSLNLSFNYHKKVSFAHLHLAPSFG
HLRSLKEL DMHG I FFRSL SE TT LQP LVQL PMLQTLRLQMNF INQAQL S I FGAF PGL LYVDL S
DNRI SGAARP
VAITREVDGRERVWL PSRNLAPRPLDTLRSEDFMPNCKAFSFTLDLSRNNLVT IQSEMFARL SRLECLRL SH
NS I SQAVNGSQFVPLTSLRVLDLSHNKLDLYHGRSFTELPRLEALDL SYNSQP FTMQGVGHNL SFVAQL PAL
RYLSLAHNDIHSRVSQQLCSASLCALDFSGNDLSRMWAEGDLYLRFFQGLRSLVWLDL SQNHLHTLL PRALD
NL PKSLKHLHLRDNNLAFFNWS SLTLLPKLETLDLAGNQLKALSNGSLPSGTQLRRLDL SGNS I GFVNPGFF
ALAKQLEELNLSANALKTVEPSWFGSMVGNLKVLDVSANPLHCACGATFVGFLLEVQAAVPGLPSRVKCGSP
GQLQGHS I FAQDLRLCLDET L SWNCFGI SLLAMALGLVVPMLHHLCGWDLWYCFHLCLAWLPHRGQRRGADA
LFYDAFVVFDKAQSAVADWVYNELRVQLEERRGRRALRLCLEERDWL PGKTLFENLWASVYS SRKTLFVLAH
TDRVSGLLRASFLLAQQRLLEDRKDVVVLVILRPDAYRSRYVRLRQRLCRQSVLLWPHQPRGQGSFWAQLGT
AL TRDNHHFYNRNFCRGP TTAE .
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The translated sequence was aligned with the four porcine TLR9 full length
cDNA sequences
deposited in Genbank (September 2011, P1-181109-prot.pro, pig-TLR9-
NM_213958.pro, pig-
TLR9-AK349013.pro, pig-TLR9-GU138029.pro, pig-TLR9-AY859728.pro). Alignment
(ClustalW; DNAStar) of the five porcine TLR9 polypeptide sequences showed
polymorphisms at
9 positions. In each case the translated sequence of our TLR9 clone (P1-181109-
prot ) conformed
to the majority polypeptide sequence, and was identical to the translation of
cDNA clone
AY859728.
Therefore, it is concluded that a correct version of porine TLR9 has been
cloned.
Example 3
Gene cloning of canine toll-like receptor 9 (TLR-9)
Total RNA from canine lymph nodes and canine spleen was purchased from Zyagen
and
was used as source for TLR9 messenger RNA (mRNA). From the canine spleen or
lymph node
total RNA, first strand cDNA was synthesized, essentially as described by the
supplier of the
reverse transcriptase ( Expand Reverse Transciptase, Roche).
Primer were designed for the polymerase chain reaction (PCR) amplification of
canine
TLR9 (Genbank NM 001002998) from the start codon region to the stop codon
(Canis-TLR9-for
and Canis-TLR9-rev, see below). However, initial PCR experiments using canine
lymph node
and spleen first strand cDNA aiming at the amplification of the full length
product (expected: ¨
3100 bp) proved to be repeatedly negative. Therefore, it was decided to
prepare two overlapping
TLR9 gene sections, that were more amenable for a PCR approach, in preparation
for a PCR
overlap extension approach. To this end primers were designed to yield a 5'-
PCR canine TLR9
product with ¨ 1600 bp (Canis-TLR9-for and CanTLR9o1r, see below), and a 3'-
PCR canine
TLR9 product with ¨ 1700 bp (CanTLR9olf and Canis-TLR9-rev).
Primer sequences:
Canis-TL R9-for GAAGCTTACCATGGGCCCCTGCCGTGGCGCCCTGCA
Canis-TLR9-rev GTCTAGATGATCAGGCTGTCGTGGGGCCCCGGCAGA
CanTLR9olf TCACCTTGGACCTGTCTCGGAACAACC
CanTLR9olr ACAGGTCCAGCTTGTTATGGGACAGG
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PCR reactions (Expand High Fidelity PCR kit, Roche) were performed, the
corresponding PCR products were agarose gel-purified, cloned into pCR2.1-Topo
(Invitrogen),
and three clones each were sequenced to identify PCR error-free versions, by
comparing their
translation products to each other, and to the database sequences NM 00102998
and AY859723.
5 These (pCR2.1-Topo-canTLR9-Nterm and pCR2.1-Topo-canTLR9-Cterm ) were
then used to
join the 5' and the 3'region of the canine TLR9 gene by an overlap extension
approach. To this
end inserts of pCR2.1-Topo-canTLR9-Nterm (primer: Canis-TLR9-for and
CanTLR9olr ) and
pCR2.1-Topo-canTLR9-Cterm (primer: CanTLR9olf and Canis-TLR9-rev) were PCR
amplified
with only 9 cycles and a proof-reading polymerase (Phusion0 Hot Start High-
Fidelity DNA
10 Polymerase, Thermo Scientific). The resulting PCR product were agarose
gel purified, and then
used combined in an overlap extension-PCR combination using the primer Canis-
TLR9-for and
Canis-TLR9-rev. The resulting ¨ 3100 bp PCR product was agarose gel purified
and cloned into
pCRBlunt-II (Invitrogen). Four independent clones were sequenced and one clone
was chosen
for further processing (pCR2.1-Topo-canineTLR9).
15 From this construct the insert was excised by HindIII/XbaI digestion,
agarose gel-
purified and subcloned into the HindIII/XbaI-cut mammalian expression vectors
pcDNA3.1(neo)
and pcDNA3.1(hyg) (both Invitrogen), yielding pcDNA3.1(neo)-canineTLR9 and
pcDNA3.1(hyg)-canineTLR9, respectively. The corresponding inserts were
resequenced (see
below).
pcDNA3.1 insert sequence canine TLR9 (primer sequences underlined, start/stop
codons
highlighted bold)
AAGCTTACCATGGGCCCCTGCCGTGGCGCCCTGCACCCCCTGTCTCTCCTGGTGCAGGCTGCCGCGCTAGC
CCTGGCCCTGGCCCAGGGCACCCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCATGGCCTGGTGAACT
GCAACTGGCTGTTCCTCAAGTCCGTGCCCCGCTTCTCGGCAGCTGCACCCCGCGGTAACGTCACCAGCCTT
TCCTTGTACTCCAACCGCATCCACCACCTCCATGACTATGACTTTGTCCACTTCGTCCACCTGCGGCGTCT
CAATCTCAAGTGGAACTGCCCGCCCGCCAGCCTCAGCCCCATGCACTTTCCCTGTCACATGACCATTGAGC
CCAACACCTTCCTGGCTGTGCCCACCCTAGAGGACCTGAATCTGAGCTATAACAGCATCACGACTGTGCCC
GCCCTGCCCAGTTCGCTTGTGTCCCTGTCCCTGAGCCGCACCAACATCCTGGTGCTGGACCCTGCCACCCT
GGCAGGCCTTTATGCCCTGCGCTTCCTGTTCCTGGATGGCAACTGCTACTACAAGAACCCCTGCCAGCAGG
CCCTGCAGGTGGCCCCAGGTGCCCTCCTGGGCCTGGGCAACCTCACACACCTGTCACTCAAGTACAACAAC
CTCACCGTGGTGCCGCGGGGCCTGCCCCCCAGCCTGGAGTACCTGCTCTTGTCCTACAACCACATCATCAC
CCTGGCACCTGAGGACCTGGCCAATCTGACTGCCCTGCGTGTCCTCGATGTGGGTGGGAACTGTCGCCGCT
GTGACCATGCCCGTAACCCCTGCAGGGAGTGCCCCAAGGGCTTCCCCCAGCTGCACCCCAACACCTTCGGC
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CACCTGAGCCACCTCGAAGGCCTGGTGTTGAGGGACAGCTCTCTCTACAGCCTGGACCCCAGGTGGTTCCA
TGGCCTGGGCAACCTCATGGTGCTGGACCTGAGTGAGAACTTCCTGTATGACTGCATCACCAAAACCAAAG
CC TTC TACGGCC TGGCCCGGCTGCGCAGACTCAACCTGTCCT TCAAT TATCATAAGAAGGTGTCCT TTGCC
CACCTGCATCTGGCATCCTCCTTCGGGAGCCTACTGTCCCTGCAGGAGCTGGACATACATGGCATCTTCTT
CCGCTCGCTCAGCGAGACCACGCTCCAGTCGCTGGCCCACCTGCCCATGCTCCAGCGTCTGCATCTGCAGT
TGAACTTTATCAGCCAGGCCCAGCTCAGCATCTTCGGCGCCTTCCCTGGCCTGCGGTACGTGGACTTGTCA
GACAACCGCATCAGTGGAGCTGCAGAGCCCGCGGCTGCCACAGGGGAGGTAGAGGCGGACTGTGGGGAGAG
AGTCTGGCCACAGTCCCGGGACCTTGCTCTGGGCACACTGGGCACCCCCGGCTCAGAGGCCTTCATGCCGA
GC TGCAGGACCC TCAACT TCACCTTGGACCTGTCTCGGAACAACC TAGTGACTGTTCAGCCGGAGATGT TT
GTCCGGCTGGCGCGCCTCCAGTGCCTGGGCCTGAGCCACAACAGCATCTCGCAGGCGGTCAATGGCTCGCA
GT TCGTGCCTCTGAGCAACC TGCGGGTGCTGGACC TGTCCCATAACAAGC TGGACC TGTACCACGGGCGCT
CGTTCACGGAGCTGCCGCGGCTGGAGGCCTTGGACCTCAGCTACAACAGCCAGCCCTTCAGCATGCGGGGC
GTGGGCCACAATCTCAGC TT TGTGGCACAGCTGCCAGCCCTGCGC TACCTCAGCCTGGCGCACAATGGCAT
CCACAGCCGCGTGTCCCAGCAGCTCCGCAGCGCCTCGCTCCGGGCCCTGGACTTCAGTGGCAATACCCTGA
GCCAGATGTGGGCCGAGGGAGACCTCTATCTCCGCTTCTTCCAAGGCCTGAGAAGCCTGGTTCAGCTGGAC
CT GTC CCAGAAT CGC C TGCATACCC TCC TGCCACGCAACC TGGACAACCT CCC CAAGAGCCT GCGGC
TCCT
GCGGCTCCGTGACAATTACCTGGCTTTCTTCAACTGGAGCAGCCTGGCCCTCCTACCCAAGCTGGAAGCCC
TGGACCTGGCGGGAAACCAGCTGAAGGCCCTGAGCAATGGCAGCTTGCCCAACGGCACCCAGCTCCAGAGG
CTGGACCTCAGCGGCAACAGCATCGGCTTCGTGGTCCCCGGC TTT TT TGCCC TGGCCGTGAGGCT TCGAGA
GC TCAACC TCAGCGCCAACGCCCTCAAGACGGTGGAGCCCTCCTGGT TTGGTTCCC TGGCGGGTGCCCTGA
AAGTCCTAGACGTGACCGCCAACCCCTTGCATTGCGCTTGCGGCGCAACCTTCGTGGACTTCTTGCTGGAG
GTGCAGGCTGCGGTGCCCGGCCTGCCTAGCCGTGTCAAGTGCGGCAGCCCGGGCCAGCTCCAGGGCCGCAG
CATCTTCGCACAGGACCTGCGCCTCTGCCTGGACGAAGCGCTCTCCTGGGTCTGTTTCAGCCTCTCGCTGC
TGGCTGTGGCCCTGAGCCTGGCTGTGCCCATGCTGCACCAGCTCTGTGGCTGGGACCTCTGGTACTGCTTC
CACCTGTGCCTGGCC TGGCTGCCCCGGCGGGGGCGGCGGCGGGGTGTGGATGCCCTGGCCTACGACGCC TT
CGTGGTCTTCGACAAGGCGCAGAGCTCGGTGGCGGACTGGGTGTACAATGAGCTGCGGGTACAGCTAGAGG
AGCGCCGTGGGCGCCGGGCGCTACGCCTGTGTCTGGAGGAACGTGACTGGGTACCCGGCAAAACCCTCTTC
GAGAACCTCTGGGCCTCAGTTTACAGCAGCCGCAAGACGCTGTTTGTGCTGGCCCGCACGGACAGAGTCAG
CGGCCTCCTGCGTGCCAGCTTCCTGCTGGCCCAACAGCGCCTGCTGGAGGACCGCAAGGACGTCGTGGTGC
TGGTGATCCTGTGCCCCGACGCCCACCGCTCCCGCTATGTGCGGCTGCGCCAGCGCCTCTGCCGCCAGAGT
GTCCTCCTCTGGCCCCACCAGCCCAGTGGCCAGCGCAGCTTCTGGGCCCAGCTGGGCACGGCCCTGACCAG
GGACAACCGCCACTTCTACAACCAGAACTTCTGCCGGGGCCCCACGACAGCCTGATCATCTA
MGPCRGALHPLSLLVQAAALALALAQGTLPAFLPCELQPHGLVNCNWLFLKSVPRFSAAAPRGNVT SLSLY
SNRIHHLHDYDFVHFVHLRRLNLKWNCP PASL SPMHFPCHMT IEPNTFLAVPTLEDLNLSYNS I TTVPALP
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sSLVSLSL SRTN I LVLDPAT LAGLYALRFLFL DGNCYYKNPCQQALQVAPGAL LGLGNLTHL SLKYNNL
TV
VPRGL P P S LEYL LL S YNH I I
TLAPEDLANLTALRVLDVGGNCRRCDHARNPCRECPKGFPQLHPNTFGHLS
HLEGLVLRDS S LY S LDPRWFHGLGNLMVL DL SENFLYDC I TKTKAFYGLARLRRLNL S
FNYHKKVSFAHLH
LAS SFGSL L SLQELD IHG I FFRSL SETT LQSLAHL PMLQRLHLQLNF I SQAQL S I
FGAFPGLRYVDL SDNR
I SGAAEPAAATGEVEADCGERVWPQ SRDLALGTLGT PGSEAFMP SCRTLNFTL DL SRNNLVTVQPEMFVRL
ARLQCLGL SHNS I SQAVNGSQFVPL SNLRVLDLSHNKLDLYHGRSFTELPRLEALDL SYNSQPFSMRGVGH
NL SFVAQL PALRYLSLAHNGIHSRVSQQLRSASLRALDFSGNTLSQMWAEGDLYLRFFQGLRSLVQLDL SQ
NRLHTLLPRNLDNLPKSLRLLRLRDNYLAFFNWSSLALLPKLEALDLAGNQLKALSNGSLPNGTQLQRLDL
SGNS I GFVVPGFFALAVRLRELNL SANALKTVE P SWFG S LAGALKVL DVTANP LHCACGATFVDFL
LEVQA
AVPGL PSRVKCGSPGQLQGRS I FAQDLRLCLDEAL SWVCFS L SL LAVAL
SLAVPMLHQLCGWDLWYCFHLC
LAWLPRRGRRRGVDALAYDAFVVFDKAQSSVADWVYNELRVQLEERRGRRALRLCLEERDWVPGKTLFENL
WASVY S SRKTLFVLARTDRVSGLLRASFLLAQQRL LEDRKDVVVLVI LCPDAHRSRYVRLRQRLCRQSVLL
WP HQ P SGQRS FWAQL GTALT RDNRHFYNQNFCRG PT TA .
The translated sequence was aligned with the two canine TLR9 full length cDNA
sequences
deposited in Genbank (September 2011, P1-010709-prot.pro, TLR-9-
NM_001002998.pro, TLR-
9-AY859723.pro). Alignment (ClustalW; DNAStar) of the three canine TLR9
polypeptide
sequences showed polymoThisms at 8 positions. Except for one position (T459 in
our sequence,
P459 in the two database sequences) all polymorphic positions in our TLR9
clone (P1-010709-
prot) conformed to either NM_00102998 or AY859723. T459 has been confirmed in
four
independent PCR products from dog lymph node and spleen cDNA suggesting that
this
corresponds to the genotype of the donor dog.
Therefore, it is concluded that a correct version of canine TLR9 has been
cloned.
Example 4
Madine-Darby canine kidney cells as NF-KB activation reporter cells
Madine-Darby canine kidney (MDCK) cells were obtained from ATCC that were
maintained in MEM, lx non-essential amino acids, 8% (v/v) iFCS. Testing of
spent growth
medium for presence of secreted alkaline phosphatase activity was negative, a
prerequisite for the
use of this cell line in reporter gene assays.
As a first step, it was planned to transfect the MDCK cells with pNifTy2-SEAP
(Invivogen), a plasmid containing a secreted alkaline phosphatase (SEAP)
reporter gene under
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the control of NF-KB binding sites, and a zeocin resistance gene as selection
marker. The
inventor's studies showed, however, that MDCK cells are largely resistant to
zeocin (up to the
mg/ml range) precluding the use of pNifTy2-SEAP. Therefore, it was decided to
generate
`pNifTy-hyg-SEAP' by replacing the CMV promoter region and parts of the
polylinker (Nru
I/Xba I digest of the plasmid and agarose gel isolation of the large fragment)
in pcDNA3.1(hyg)
by the Swa I/NheI fragment containing the NF-KB binding sites and the SEAP
gene of pNifTy2-
SEAP. Thereby, presence of the reporter gene cassette could now be selected
for by addition of
hygromycin to the medium, a cytostatic that is effective on MDCK cell.
MDCK cells were transfected with pNifTy-hyg-SEAP, selection pressure was
applied
1 0 (300 ug/m1hygromycin) and a resistant line was selected by repeated
subculture in selection
medium. The selected cell line was tested for SEAP induction by human tumor
necrosis factor
(huTNF-a, + positive control for proper functioning of the NF-KB pathway and
reporter gene
activation) as well as by a selection of pathogen-associated molecular
patterns (PAMPs, such as
E. coli lipopolysaccharide (LPS, TLR4), poly-I/polyC (double-stranded RNA,
TLR3), muramyl
1 5 dipeptide (MDP, NOD2), PAM3Cy5SK4 (a synthetic lipopeptide, TLR1/2),
and R-848 (a low
molecular weight agonist of TLR7). The results are shown in figures 1 and 2
(figure 2 is a y-axis
expansion of figure 1).
huTNF-a potently induces SEAP production in a polyclonal MDCK-pNifTy-hyg-SEAP
20 cell line, a second prerequisite for the use of this cell line in
reporter gene assays. PAMPs
addressing 5 different pattern recognition receptors feeding into the NF-KB
pathway showed low
or no SEAP induction, which suggests that our MDCK-pNifTy-hyg-SEAP cell line
expresses
none or very few copies of the corresponding receptors, a third prerequisite
for the use of this cell
line in reporter gene assays. The highest background (albeit still very low)
was seen with dsRNA,
25 followed by MDP, while LPS, PAM3Cy5SK4 and R-848 showed virtually none.
These results prompted the inventors to perform single cell cloning of the
MDCK-
pNifTy-hyg-SEAP cell line, to stabilize the properties seen in the polyclonal
line and to identify a
superior clone. 46 clones were selected by limiting dilution in 96 well
plates, and following
expansion, they were stimulated with huTNF-a to identify the clones with the
highest NF-KB-
30 induced SEAP production capacity (see figure 3).
Example 5
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Generation of canine, porcine and bovine TLR9-21 fusion constructs and
subcloning into
expression vector pIRES-puro.
A fusion construct encoding the extracellular domain of bovine TLR9 and
intracellular domain of
chicken TLR21 was created using a "PCR-sewing" protocol.
"PCR-sewing" entails three steps (see figure 4): first, complementary
sequences are added by
PCR to DNA fragments that should be fused into one construct. Secondly, the
two fragments
having complementary sequences are combined in a PCR-reaction without addition
of primers.
The complementary sequences enable the fragments to anneal and prime the
elongation reaction
by DNA polymerase. In the third PCR step, primers annealing to the 5' and 3'
end of the
chimeric molecule are added to amplify the fusion molecule.
The sequence encoding the extracellular domain of bovine TLR9 was amplified
from the
pcDNA3.1(neo)-bovine TLR9 construct (section 1) by PCR, the sequence encoding
the
transmembrane and intracellular domain of chicken TLR21 was amplified from
pcDNA3.1(neo)-
chicken TLR21 (sequences below). Complementary sequences were added to the
3'end of the
extracellular (TLR9) fragment and the 5' end of the TLR21 fragment using
primers with a 5'
overhang (sequences below) by PCR. Expand High Fidelity PCR kit (Roche) was
used for all
PCRs.
pcDNA3.1(neo)-chicken TLR21 insert sequence, start/stop codons highlighted
bold.
AAGCTTACCATGATGGAGACAGCGGAGAAGGCATGGCCCAGCACCAGGATGTGCCCCTCCCACTGCTGTCC
ACTCTGGCTGCTGCTGCTGGTGACAGTGACACTGATGCCGATGGTGCACCCGTATGGCTTTCGCAACTGCA
TTGAGGATGTCAAGGCACCTTTGTACTTCCGCTGCATCCAGCGCTTCCTGCAGTCGCCGGCCCTGGCAGTG
TCTGACCTGCCACCACATGCCATCGCGCTCAATCTGTCATACAACAAAATGCGCTGCCTGCAGCCCTCTGC
CTTTGCCCACCTGACACAGCTGCATACCCTGGACCTGACCTACAACCTCCTGGAGACCCTCTCCCCTGGTG
CCTTCAATGGGCTGGGTGTGCTGGTGGTGCTGGACCTGTCTCACAACAAGCTGACCACACTTGCTGAAGGG
GTGTTCAACAGCTTGGGCAACCTGTCCTCGCTGCAGGTACAACATAACCCCCTCAGCACGGTGTCACCAAG
TGCTCTGCTACCCCTGGTCAACCTGCGCCGCCTGTCTCTACGGGGCGGGCGGCTGAATGGGTTGGGGGCAG
TGGCAGTGGCAGTGCAGGGCTTGGCACAGCTGGAGCTGTTGGACCTATGTGAAAACAACCTGACAACGCTG
GGGCCAGGCCCACCGCTACCCGCCTCGCTGCTCACCCTGCAGCTGTGCAACAACTCGCTGAGGGAGTTAGC
GGGGGGCAGCCCGGAGATGCTATGGCACGTGAAGATACTCGACCTCTCCTACAACAGTATCTCACAGGCGG
AGGTCTTCACCCAGCTCCACCTGCGCAACATCAGCCTGCTCCACCTGATCGGCAACCCCTTGGATGTCTTC
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CACCTGTTGGACATCTCTGACATCCAACCTCGCAGCCTGGATTTCTCTGGGTTGGTGCTGGGGGCTCAGGG
GCTGGATAAGGTGTGCCTGAGGCTGCAGGGTCCCCAGGCCTTGCGGCGGCTGCAGCTACAACGCAACGGGC
TGAAGGTGCTGCATTGTAATGCACTGCAGTTGTGTCCTGTGCTGAGAGAGCTGGACCTGTCCTGGAACCGG
CTACAGCACGTGGGCTGTGCCGGCCGGCTGCTGGGCAAGAAGCAGCGGGAGAAGCTGGAAGTGCTGACAGT
5 GGAACACAACCTGCTGAAGAAACTGCCGTCTTGCCTGGGGGCCCAGGTGCTGCCTCGGCTGTACAACATTT
CCTTCCGCTTTAACCGCATCCTGACTGTTGGGCCCCAAGCCTTTGCCTACGCCCCGGCCCTGCAGGTGTTG
TGGCTCAATATTAACAGCCTGGTGTGGCTGGACAGGCAGGCACTGTGGAGGCTGCACAACCTGACAGAGCT
GCGCCTGGACAACAACCTGCTGACCGACCTCTATCACAACTCCTTCATTGACCTCCACAGACTGCGCACCC
TCAACCTGCGCAACAACCGTGTCTCCGTCCTCTTCTCTGGTGTCTTCCAGGGGCTGGCTGAGCTGCAGACG
10 CTGGATTTAGGGGGCAACAACTTGCGCCACCTGACTGCACAGTCACTGCAGGGGCTGCCCAAACTGCGCAG
GCTGTACCTGGACCGCAACAGATTGCTGGAGGTGAGCAGCACTGTGTTCGCCCCAGTGCAGGCTACCCTGG
GGGTGCTGGACCTGCGGGCCAACAACCTGCAGTACATCTCACAGTGGCTGCGCAAGCCGCCACCCTTCCGC
AACCTGAGCAGCCTGTACGACCTGAAGCTGCAGGCGCAGCAGCCCTATGGACTGAAGATGCTGCCTCACTA
CTTCTTCCAGGGCTTGGTGAGGCTGCAGCAGCTGTCGCTGTCACAGAACATGCTGCGGTCCATCCCACCGG
15 ATGTCTTCGAGGACTTGGGCCAGCTGCGCTCCCTGGCATTGGCTGACAGCAGCAATGGGCTGCATGACCTG
CCTGACGGCATCTTCAGAAACCTGGGCAACCTGCGGTTCCTGGACCTGGAGAATGCAGGGCTGCACTCGCT
CACTCTGGAAGTCTTCGGCAATCTCAGCCGGCTGCAGGTGCTGCACTTGGCCAGAAACGAGCTGAAGACCT
TCAATGACAGCGTTGCCAGCCGGCTGTCCTCCTTGCGCTACCTGGACCTGCGCAAGTGTCCGCTCAGCTGC
ACCTGTGACAACATGTGGCTGCAGGGCTGGCTGAACAACAGCCGTGTGCAGGTTGTCTACCCCTACAACTA
20 CACCTGTGGCTCACAGCACAATGCCTACATCCACAGCTTTGACACACACGTCTGCTTCCTGGACCTGGGGC
TCTATCTCTTTGCTGGGACTGCACCGGCAGTGCTGCTGCTGCTGGTGGTGCCGGTGGTGTACCACCGCGCC
TACTGGAGGCTGAAGTACCACTGGTACCTTCTGCGGTGCTGGGTCAACCAGCGGTGGCGGCGGGAGGAAAA
GTGCTACCTCTATGACAGCTTTGTGTCCTACAATTCAGCTGATGAAAGTTGGGTGTTGCAGAAGCTGGTGC
CTGAGCTGGAGCACGGTGCCTTCCGCCTCTGCTTGCACCACCGCGACTTCCAGCCGGGCCGCAGCATCATT
25 GACAACATTGTGGATGCTGTCTACAACAGCCGGAAGACGGTGTGCGTGGTGAGCCGCAGCTACCTGCGCAG
CGAGTGGTGCTCTCTAGAGGTGCAGTTGGCCAGCTACCGGCTGTTGGATGAGCGGCGTGACATCCTGGTAC
TGGTGCTGCTGGAGGACGTGGGTGATGCTGAGCTGTCTGCCTACCACCGCATGCGGCGGGTGCTGCTGCGG
CGCACCTACCTGCGCTGGCCTCTTGACCCCGCAGCTCAGCCGCTCTTTTGGGCACGGCTGAAGAGGGCACT
GAGGTGGGGAGAGGGAGGAGAGGAGGAGGAAGAAGAAGGTTTGGGTGGAGGGACGGGAAGGCCCAGGGAAG
30 GAGACAAACAGATGTAGCGGCCGC
Primers for bovine TLR9 (extracellular domain):
cowT9 -chT21 5 'Eco: GCGGATATCACCATGGGCCCCTACTGTGC
cowT9 -chT2 1 fusRV: ATAGAGC C CCAGGTC CAGGAAGCAGAGGC GCAGGTC CT GTGT
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Primers for chicken TLR21 (transmembrane and intracellular domain):
cowT9-chT21fusFW: ACACAGGACCTGCGCCTCTGCTTCCTGGACCTGGGGCTCTAT
cowT9-chT21 3' Eco: GCGGAATTCCTACATCTGTTTGTCTCCTT.
The fusion product was cloned into pCRII-TOPO (Invitrogen) and one (1) clone
was sequenced
to examine if the sequence was PCR error-free. The sequence contained one (1)
coding mutation
which was corrected using the Quik Change II XL site directed mutagenesis kit
from Stratagene
and primers:
Mutagenesis primers:
18- CowT9-chT21:
CCAAGACCACCATCTTCAACGACCTGACCCAGCTGCGCAGACTCAACC
19- CowT9-chT21:
GGTTGAGTCTGCGCAGCTGGGTCAGGTCGTTGAAGATGGTGGTCTTGG
After the mutagenesis procedure, multiple (5) clones were sequenced to examine
if site-directed
mutagenesis had been successful and did not introduce new mutations. A correct
clone was used
to redone the fusion construct into pIRESpuro3 (Clontech) using the primer-
introduced EcoRI
and EcoRV sites. The fusion construct in the resulting vector (p1RESpuro-
bovTLR9-21) was
resequenced (see below).
pIRESpuro-bovTLR9-21 insert sequence, (partial) primer sequence underlined,
TLR21 (coding)
sequences in italics, start/stop codons highlighted bold.
GATATCACCATGGGCCCCTACTGTGCCCCGCACCCCCTTTCTCTCCTGGTGCAGGCGGCGGCACTGGCAGC
GGCCCTGGCCGAGGGCACCCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCATGGTCAGGTGGACTGCA
ACTGGCTGTTCCTGAAGTCTGTGCCGCACTTTTCGGCTGGAGCCCCCCGGGCCAATGTCACCAGCCTCTCC
TTAATCTCCAACCGCATCCACCACTTGCATGACTCTGACTTCGTCCACCTGTCCAACCTGCGGGTCCTCAA
CCTCAAGTGGAACTGCCCGCCGGCCGGCCTCAGCCCCATGCACTTCCCCTGCCGTATGACCATCGAGCCCA
ACACCTTCCTGGCTGTGCCCACCCTGGAGGAGCTGAACCTGAGCTACAACGGCATCACGACCGTGCCTGCC
CTGCCCAGTTCCCTCGTGTCCCTGTCGCTGAGCCACACCAGCATCCTGGTGCTAGGCCCCACCCACTTCAC
CGGCCTGCACGCCCTGCGCTTTCTGTACATGGACGGCAACTGCTACTACATGAACCCCTGCCCGCGGGCCC
TGGAGGTGGCCCCAGGCGCCCTCCTCGGCCTGGGCAACCTCACGCACCTGTCGCTCAAGTACAACAACCTC
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AC GGAGGT GCCCCGCCGCC TGCCCCCCAGCC TGGACACCC TGC TGC TG TCC TACAACCACAT TG
TCACCC T
GGCACCCGAGGACCTGGCCAACCTGACTGCCCTGCGCGTGCTTGACGTGGGTGGGAACTGCCGCCGCTGCG
ACCACGCCCGCAACCCCTGCAGGGAGTGCCCAAAGAACTTCCCCAAGCTGCACCCTGACACCTTCAGTCAC
CTGAGCCGCCTCGAAGGCCTGGTGTTGAAGGACAGTTCTCTCTACAAACTAGAGAAAGATTGGTTCCGCGG
CC TGGGCAGGC TCCAAGTGC TCGACC TGAGTGAGAACTTCCTCTATGACTACATCACCAAGACCACCATCT
TCAACGACCTGACCCAGCTGCGCAGACTCAACCTGTCCTTCAATTACCACAAGAAGGTGTCCTTCGCCCAC
CTGCACCTAGCGTCCTCCTTTGGGAGTCTGGTGTCCCTGGAGAAGCTGGACATGCACGGCATCTTCTTCCG
CTCCCTCACCAACATCACGCTCCAGTCGCTGACCCGGCTGCCCAAGCTCCAGAGTCTGCATCTGCAGCTGA
AC TTCATCAACCAGGCCCAGCTCAGCATC TTTGGGGCC TTCCCGAGCCTGC TCT TCGTGGACC TGTCGGAC
AACCGCATCAGCGGAGCCGCGACGCCAGCGGCCGCCCTGGGGGAGGTGGACAGCAGGGTGGAAGTCTGGCG
AT TGCCCAGGGGCC TCGCTCCAGGCCCGCTGGACGCCGTCAGCTCAAAGGACTTCATGCCAAGC TGCAACC
TCAACTTCACCTTGGACCTGTCACGGAACAACCTGGTGACAATCCAGCAAGAGATGTTTACCCGCCTCTCC
CGCCTCCAGTGCCTGCGCCTGAGCCACAACAGCATCTCGCAGGCGGTTAATGGCTCCCAGTTCGTGCCGCT
GACCAGCCTGCGAGTGCTCGACCTGTCCCACAACAAGCTGGACCTGTACCATGGGCGCTCATTCACGGAGC
TGCCGCAGCTGGAGGCACTGGACCTCAGCTACAACAGCCAGCCCTTCAGCATGCAGGGCGTGGGCCACAAC
CTCAGCTTCGTGGCCCAGCTGCCCTCCCTGCGCTACCTCAGCCTTGCGCACAATGGCATCCACAGCCGCGT
GTCACAGAAGCTCAGCAGCGCCTCGTTGCGCGCCCTGGACTTCAGCGGCAACTCCCTGAGCCAGATGTGGG
CCGAGGGAGACCTCTATCTCTGCTTTTTCAAAGGCTTGAGGAACCTGGTCCAGCTGGACCTGTCCGAGAAC
CATCTGCACACCCTCCTGCCTCGTCACCTGGACAACCTGCCCAAGAGCCTGCGGCAGCTGCGTCTCCGGGA
CAATAACCTGGCCTTCTTCAACTGGAGCAGCCTGACCGTCCTGCCCCGGCTGGAAGCCCTGGATCTGGCAG
GAAACCAGCTGAAGGCCCTGAGCAACGGCAGCCTGCCGCCTGGCATCCGGCTCCAGAAGCTGGACGTGAGC
AGCAACAGCATCGGCTTCGTGATCCCCGGCTTCTTCGTCCGCGCGACTCGGCTGATAGAGCTTAACCTCAG
CGCCAATGCCCTGAAGACAGTGGATCCCTCCTGGTTCGGTTCCTTAGCAGGGACCCTGAAAATCCTAGACG
TGAGCGCCAACCCGCTCCACTGCGCCTGCGGGGCGGCCTTTGTGGACTTCCTGCTGGAGAGACAGGAGGCC
GTGCCCGGGCTGTCCAGGCGCGTCACATGTGGCAGTCCGGGCCAGCTCCAGGGCCGCAGCATCTTCACACA
GGACCTGCGCCTCTGC TTCCTGGACCTGGGGCTCTATCTCTTTGCTGGGACTGCACCGGCAGTGCTGCTGC
TGCTGGTGGTGCCGGTGGTGTACCACCGCGCCTACTGGAGGCTGAAGTACCACTGGTACCTTCTGCGGTGC
TGGGTCAACCAGCGGTGGCGGCGGGAGGAAAAGTGCTACCTCTATGACAGCTTTGTGTCCTACAATTCAGC
TGATGAAAGTTGGGTGTTGCAGAAGCTGGTGCCTGAGCTGGAGCACGGTGCCTTCCGCCTCTGCTTGCACC
ACCGCGACTTCCAGCCGGGCCGCAGCATCATTGACAACATTGTGGATGCTGTCTACAACAGCCGGAAGACG
GTGTGCGTGGTGAGCCGCAGCTACCTGCGCAGCGAGTGGTGCTCTCTAGAGGTGCAGTTGGCCAGCTACCG
GCTGTTGGATGAGCGGCGTGACATCCTGGTACTGGTGCTGCTGGAGGACGTGGGTGATGCTGAGCTGTCTG
CCTACCACCGCATGCGGCGGGTGCTGCTGCGGCGCACCTACCTGCGCTGGCCTCTTGACCCCGCAGCTCAG
CCGCTCTTTTGGGCACGGCTGAAGAGGGCACTGAGGTGGGGAGAGGGAGGAGAGGAGGAGGAAGAAGAAGG
TTTGGGTGGAGGGACGGGAAGGCCCAGGGAAGGAGACAAACAGA TGTAGGAAT TC
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MGPYCAPHPL S LLVQAAALAAALAEGT L PAFL PCELQPHGQVDCNWLFLKSVPHFSAGAPRANVT SL SL
IS
NRIHHLHDSDFVHL SNLRVLNLKWNCP PAGL S PMHF PCRMT I EPNTFLAVP TLEELNL
SYNGITTVPALP S
SLVSL SLSHTS I LVLGP THFTGLHALRFLYMDGNCYYMNPC PRALEVAPGALLGLGNLTHL SLKYNNLTEV
PRRL P P SL DTL LL S YNH IVTLAPEDLANLTALRVLDVGGNCRRCDHARNPCREC PKNF
PKLHPDTFSHL SR
LEGLVLKDSSLYKLEKDWFRGLGRLQVLDL SENFLYDY I TKT T I FNDL TQLRRLNL
SFNYHKKVSFAHLHL
AS SFGSLVSLEKLDMHG I FFRSLTNI T LQS LTRL PKLQS LHLQLNFINQAQL S I FGAF P SL
LFVDL S DNRI
SGAATPAAALGEVDSRVEVWRLPRGLAPGPLDAVS SKDFMPSCNLNFTLDL SRNNLVTIQQEMFTRL SRLQ
CLRLSHNS I SQAVNGSQFVPLTSLRVLDLSHNKLDLYHGRSFTEL PQLEALDLSYNSQPFSMQGVGHNLSF
VAQLP SLRYLSLAHNGIHSRVSQKLSSASLRALDFSGNSLSQMWAEGDLYLCFFKGLRNLVQLDLSENHLH
1 0 TLLPRHLDNLPKSLRQLRLRDNNLAFFNWSSLTVL PRLEALDLAGNQLKALSNGSL P PG
IRLQKLDVS SNS
IGFVI PGFFVRATRL IELNLSANALKTVDP SWFGSLAGT LK I LDVSANPLHCACGAAFVDFLLERQEAVPG
LSRRVTCGSPGQLQGRS I FTQDLRLC FL DLGL YL FAGTAPAVLLLLVVPVVY HRAYWRLKYHWY
LLRCWVN
QRWRREEKCYL Y DS FVS YNSADESWVLQKLVPELEHGAFRI,CLHHRDFQPGRS I I DNI VDAVYNS
RKTVCV
VS R S YLR S EWCS LEVQLA S YR LLDERRD ILVI,VI,LEDVGDAEL S AYHRMRRVI,LRRT
YLRWPI, DPAAQPI, F
WARLKRALRWGEGGEEEEEEGLGGGTGRPREGDKQM
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Cloning of a fusion construct of the extracellular domain of porcine TLR9 and
the
transmembrane and intracellular domain of chicken TLR21.
A fusion construct encoding the extracellular domain of porcine TLR9 and
intracellular domain
of chicken TLR21 was created using the "PCR-sewing" protocol as described
above.
The sequence encoding the extracellular domain of porcine TLR9 from the
pcDNA3.1(neo)-
porcine TLR9 construct (described in Eample 2) was amplified by PCR as well as
the sequence
encoding the transmembrane and intracellular domain of chicken TLR21 from
pcDNA3.1(neo)-
chicken TLR21 (sequences above). Complementary sequences were added to the 3
'end of the
extracellular (TLR9) fragment and the 5'end of the TLR21 fragment using
primers with a 5'
overhang (sequences below) by PCR. Expand High Fidelity PCR kit (Roche) was
used for all
PCRs.
Primers for porcine TLR9 (extracellular domain):
piT9-chT21 5' E: GCGGAATTCCACCATGGGCCCCCGCTGCAC
pigT9-chT21fusRV: ATAGAGCCCCAGGTCCAGGAAGCAGAGGCGCAGGTCTTGCGC
Primers for chicken TLR21 (transmembrane and intracellular domain):
pigT9-chT21fusFW: GCGCAAGACCTGCGCCTCTGCTTCCTGGACCTGGGGCTCTAT
pig/dogT9-chT21-: GCGGCGGCCGCCTACATCTGTTTGTCTCCTT
The fusion product was cloned into pCRII-TOPO (Invitrogen) and one (1) clone
was sequenced
to examine if the sequence was PCR error-free. This clone was correct and was
used to redone
the fusion construct into pIRESpuro3 (Clontech) using primer-introduced EcoRI
and NotI sites.
The 5' and 3' ligation site of the fusion construct in pIRESpuro3 were
sequenced to check the
correct insertion of the fragment in the plasmid. The correct, resulting
vector is pIRESpuro-
porTLR9-21 (sequences below).
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pIRESpuro-porTLR9-21 insert sequence, (partial) primer sequence underlined,
TLR21 (coding)
sequences in italics, start/stop codons highlighted bold.
GAATTCCACCATGGGCCCCCGCTGCACCCTGCACCCCCTTTCTCTCCTGGTGCAGGTGACAGCGCTGGCTG
5 CGGCTCTGGCCCAGGGCAGGCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCACGGCCTGGTGAACTGC
AACTGGCTCTTCCTGAAGTCCGTGCCCCACTTCTCGGCGGCAGCGCCCCGGGCCAACGTCACCAGCCTCTC
CT TACTCTCCAACCGCATCCACCACCTGCACGACTCTGACTTCGTCCACCTGTCCAGCCTACGAACTCTCA
ACCTCAAGTGGAACTGCCCGCCGGCTGGCCTCAGCCCCATGCACTTCCCCTGCCACATGACCATCGAGCCC
AACACCTTCCTGGCCGTGCCCACCCTGGAGGAGCTGAACCTGAGCTACAACAGCATCACGACCGTGCCTGC
10 CCTGCCCGACTCCCTCGTGTCCCTGTCGCTGAGCCGCACCAACATCCTGGTGCTAGACCCCACCCACCTCA
CTGGCCTACATGCCCTGCGCTACCTGTACATGGATGGCAACTGCTACTACAAGAACCCCTGCCAGGGGGCG
CTGGAGGTGGTGCCGGGTGCCCTCCTCGGCCTGGGCAACCTCACACATCTCTCACTCAAGTACAACAATCT
CACGGAGGTGCCCCGCAGCCTGCCCCCCAGCCTGGAGACCCTGCTGTTGTCCTACAACCACATTGTCACCC
TGACGCCTGAGGACCTGGCCAATCTGACTGCCCTGCGCGTGCTTGATGTGGGGGGGAACTGCCGCCGCTGT
15 GACCATGCCCGCAACCCCTGCAGGGAGTGCCCAAAGGACCACCCCAAGCTGCACTCTGACACCTTCAGCCA
CCTGAGCCGCCTCGAAGGCCTGGTGTTGAAAGACAGTTCTCTCTACAACCTGGACACCAGGTGGTTCCGAG
GCCTGGACAGGCTCCAAGTGCTGGACCTGAGTGAGAACTTCCTCTACGACTGCATCACCAAGACCACGGCC
TTCCAGGGCCTGGCCCGACTGCGCAGCCTCAACCTGTCCTTCAATTACCACAAGAAGGTGTCCTTTGCCCA
CCTGCACCTGGCACCCTCCTTTGGGCACCTCCGGTCCCTGAAGGAGCTGGACATGCATGGCATCTTCTTCC
20 GCTCGCTCAGTGAGACCACGCTCCAACCTCTGGTCCAACTGCCTATGCTCCAGACCCTGCGCCTGCAGATG
AACTTCATTAACCAGGCCCAGCTCAGCATCTTTGGGGCCTTCCCTGGCCTGCTGTACGTGGACCTATCGGA
CAACCGCATCAGCGGAGCTGCAAGGCCAGTGGCCATTACTAGGGAGGTGGATGGTAGGGAGAGGGTCTGGC
TGCCTTCCAGGAACCTCGCTCCACGTCCACTGGACACTCTCCGCTCAGAGGACTTCATGCCAAACTGCAAG
GCCTTCAGCTTCACCTTGGACCTGTCTCGGAACAACCTGGTGACAATCCAGTCGGAGATGTTTGCTCGCCT
25 CTCACGCCTCGAGTGCCTGCGCCTGAGCCACAACAGCATCTCCCAGGCGGTCAATGGCTCTCAGTTTGTGC
CGCTGACCAGCCTGCGGGTGCTGGACCTGTCCCACAACAAGCTGGACCTGTATCACGGGCGCTCGTTCACG
GAGCTGCCGCGCCTGGAAGCACTGGACCTCAGCTACAATAGCCAGCCCTTTACCATGCAGGGTGTGGGCCA
CAACCTCAGCTTCGTGGCCCAGCTGCCCGCCCTGCGCTACCTCAGCCTGGCGCACAATGACATCCATAGCC
GAGTGTCCCAGCAGCTCTGTAGCGCCTCACTGTGCGCCCTGGACTTTAGCGGCAACGATCTGAGCCGGATG
30 TGGGCTGAGGGAGACCTCTATCTCCGCTTCTTCCAAGGCCTAAGAAGCCTAGTCTGGCTGGACCTGTCCCA
GAACCACCTGCACACCCTCCTGCCACGTGCCCTGGACAACCTCCCCAAAAGCCTGAAGCATCTGCATCTCC
GTGACAATAACCTGGCCTTCTTCAACTGGAGCAGCCTGACCCTCCTGCCCAAGCTGGAAACCCTGGACTTG
GC TGGAAACCAGCTGAAGGCCCTAAGCAATGGCAGCCTGCCATCTGGCACCCAGCTGCGGAGGC TGGACC T
CAGTGGCAACAGCATCGGCTTTGTGAACCCTGGCTTCTTTGCCCTGGCCAAGCAGTTAGAAGAGCTCAACC
35 TCAGCGCCAATGCCCTCAAGACAGTGGAGCCCTCCTGGTTTGGCTCGATGGTGGGCAACCTGAAAGTCCTA
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GACGTGAGCGCCAACCCTCTGCACTGTGCCTGTGGGGCGACCTTCGTGGGCTTCCTGCTGGAGGTACAGGC
TGCCGTGCCTGGGCTGCCCAGCCGCGTCAAGTGTGGCAGTCCGGGGCAGCTCCAGGGCCATAGCATCTTTG
CGCAAGACCTGCGCCTCTGC TT CCT GGACCTGGGGCT C TAT CT CTTTGCTGGGACTGCACCGGCAGTGCTG
CTGCTGCTGGTGGTGCCGGTGGTGTACCACCGCGCCTACTGGAGGCTGAAGTACCACTGGTACCTTCTGCG
GT GCTGGGT CAACCAGCGGTGGCGGCGGGAGGAAAAGTGCTACCT CTATGACAGCTTT GTGT CCTACAAT T
CAGCTGATGAAAGTTGGGTGTTGCAGAAGCTGGTGCCTGAGCTGGAGCACGGTGCCTTCCGCCTCTGCTTG
CACCACCGCGACTTCCAGCCGGGCCGCAGCATCATTGACAACATTGTGGATGCTGTCTACAACAGCCGGAA
GACGGTGT GCGTGGTGAGCCGCAGCTACCT GCGCAGCGAGTGGTGCTCT CTAGAGGTGCAGTTGGCCAGCT
ACCGGCTGTTGGAT GAGCGGCGTGACAT CC TGGTACTGGTGCTGC TGGAGGACGTGGGTGATGCTGAGCTG
TCTGCCTACCACCGCATGCGGCGGGTGCTGCTGCGGCGCACCTACCTGCGCTGGCCTCTTGACCCCGCAGC
T CAGCCGCT CT TTTGGGCACGGCT GAAGAGGGCACTGAGGTGGGGAGAGGGAGGAGAGGAGGAGGAAGAAG
AAGGTTTGGGTGGAGGGACGGGAAGGCCCAGGGAAGGAGACAAACAGATGTAGGC GGCC GC
MGPRCTLHPLSLLVQVTALAAALAQGRLPAFLPCELQPHGLVNCNWLFLKSVPHFSAAAPRANVTSL SLL S
NRIHHLHDSDFVHLSSLRTLNLKWNC PPAGLS PMHFPCHMT I EPNTFLAVP TLEELNL SYNS I
TTVPALPD
SLVSL SL SRTN I LVLDP THL TGLHALRYLYMDGNCYYKNPCQGALEVVPGALLGLGNL THL
SLKYNNLTEV
PRSLP PSLETLLLSYNHIVTL TPEDLANLTALRVLDVGGNCRRCDHARNPCREC PKDHPKLHSDTFSHL SR
LEGLVLKDSSLYNLDTRWFRGLDRLQVLDLSENFLYDC I TKTTAFQGLARLRSLNL SFNYHKKVSFAHLHL
AP SFGHLRSLKELDMHG I FFRSL SETT LQP LVQL PMLQT LRLQMNF INQAQL S I FGAF PGL
LYVDL S DNR I
SGAARPVA I TREVDGRERVWL PSRNLAPRPLDTLRSEDFMPNCKAFSFTLDLSRNNLVT I Q SEMFARL SRL
EC LRL SHNS I SQAVNGSQFVPLTSLRVLDL SHNKLDLYHGRSFTELPRLEALDL SYNSQPFTMQGVGHNL S
FVAQL PALRYLSLAHNDIHSRVSQQLCSASLCALDFSGNDL SRMWAEGDLYLRFFQGLRSLVWLDL SQNHL
HT LL PRAL DNL PKSLKHLHLRDNNLAFFNWSSL TLL PKLETLDLAGNQLKALSNGSLP SGTQLRRLDLSGN
S I GFVNPGFFALAKQLEELNL SANALKTVE P SWFGSMVGNLKVLDVSANPLHCACGAT FVGFLLEVQAAVP
GL PSRVKCGSPGQLQGHS I FAQDLRLCFLDLGLYLFAGTAPAVLLLLVVPVVYHRAYWRLKYHWYLLRCWV
NQRWRREEKCYLY DSFVS YNS ADE SWVLQKLVPELEHGAFRLCLHHRDFQPGRS I IDNIVDAVYNSRKTVC
VVSRS YLR SEW CSLEVQLAS Y RLL DERRDI LVLVLLEDVGDAEL S AY HRMRRVLLRRT YLRW PL
DPAAQ PL
FWARLKRALRWGEGGEEEEEEGLGGGTGRPREGDKQM
Cloning of a fusion construct of the extracellular domain of canine TLR9 and
the
transmembrane and intracellular domain of chicken TLR21.
A fusion construct encoding the extracellular domain of canine TLR9 and
intracellular domain of
chicken TLR21 was created using the "PCR-sewing" protocol as described above.
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The sequence encoding the extracellular domain of canine TLR9 from the
pcDNA3.1(neo)-
canine TLR9 construct (described in example 3) was amplified by PCR as well as
the sequence
encoding the transmembrane and intracellular domain of chicken TLR21 from
pcDNA3.1(neo)-
chicken TLR21 (sequences above). Complementary sequences were added to the 3
'end of the
extracellular (TLR9) fragment and the 5'end of the TLR21 fragment using
primers with a 5'
overhang (sequences below) by PCR. Expand High Fidelity PCR kit (Roche) was
used for all
PCRs.
Primers for canine TLR9 (extracellular domain):
doT9-chT21 5' E: GCGGAATTCCACCATGGGCCCCTGCCGTGG
dogT9 -chT2 1 fusRV: ATAGAGC C C CAGGTC CAGGAAGCAGAGGC GCAGGT C CT GTGC
Primers for chicken TLR21 (transmembrane and intracellular domain):
dogT9 -chT2 1 fusFW: GCACAGGAC CT GC GC CT CT GCTTC CTGGAC CTGGGGCTCTAT
pig/dogT9-chT21-: GCGGCGGCCGCCTACATCTGTTTGTCTCCTT
The fusion product was cloned into pCRII-TOPO (Invitrogen) and one clone was
sequenced to
examine if the sequence was PCR error-free. The sequence contained one coding
and one silent
mutation. The coding mutation was corrected using the Quik Change II XL site
directed
mutagenesis kit (Stratagene) and primers:
Mutagenesis primers:
dT9-chT21mt FW: GCAGGCTGCCGCGCTAGCCCTGGCCCTGGCCCAGGGC
dT9-chT21mt RV: GCCCTGGGCCAGGGCCAGGGCTAGCGCGGCAGCCTGC
After the mutagenesis procedure, multiple (8) clones were sequenced to examine
if site-directed
mutagenesis had been successful. One clone contained the corrected nucleotide.
This clone was
used to redone the fusion construct into pIRESpuro3 (Clontech) using the
primer-introduced
EcoRI and the EcoRI site present in pCRII-TOPO. The resulting vector
(pIRESpuro-canTLR9-
21) was completely sequenced. Sequencing identified two silent mutations which
do not affect
the amino acid sequence. Therefore it was concluded that this clone could be
used for further
applications.
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pIRESpuro-canTLR9-21 insert sequence, (partial) primer sequence underlined,
TLR21 (coding)
sequences in italics, start/stop codons highlighted bold.
GAATCCACCATGGGCCCCTGCCGTGGCGCCCTGCACCCCCTGTCTCTCCTGGTGCAGGCTGCCGCGCTAGC
CCTGGCCCTGGCCCAGGGCACCCTGCCTGCCTTCCTGCCCTGTGAGCTCCAGCCCCATGGCCTGGTGAACT
GCAACTGGCTGTTCCTCAAGTCCGTGCCCCGCTTCTCGGCAGCTGCACCCCGCGGTAACGTCACCAGCCTT
TCCTTGTACTCCAACCGCATCCACCACCTCCATGACTATGACTTTGTCCACTTCGTCCACCTGCGGCGTCT
CAATCTCAAGTGGAACTGCCCGCCCGCCAGCCTCAGCCCCATGCACTTTCCCTGTCACATGACCATTGAGC
CCAACACCTTCCTGGCTGTGCCCACCCTAGAGGACCTGAATCTGAGCTATAACAGCATCACGACTGTGCCC
GCCCTGCCCAGTTCGCTTGTGTCCCTGTCCCTGAGCCGCACCAACATCCTGGTGCTGGACCCTGCCACCCT
GGCAGGCCTTTATGCCCTGCGCTTCCTGTTCCTGGATGGCAACTGCTACTACAAGAACCCCTGCCAGCAGG
CCCTGCAGGTGGCCCCAGGTGCCCTCCTGGGCCTGGGCAACCTCACACACCTGTCACTCAAGTACAACAAC
CTCACCGTGGTGCCGCGGGGCCTGCCCCCCAGCCTGGAGTACCTGCTCTTGTCCTACAACCACATCATCAC
CCTGGCACCTGAGGACCTGGCCAATCTGACTGCCCTGCGTGTCCTCGATGTGGGTGGGAACTGTCGCCGCT
GTGACCATGCCCGTAACCCCTGCAGGGAGTGCCCCAAGGGCTTCCCCCAGCTGCACCCCAACACCTTCGGC
CACCTGAGCCACCTCGAAGGCCTGGTGTTGAGGGACAGCTCTCTCTACAGCCTGGACCCCAGGTGGTTCCA
TGGCCTGGGCAACCTCATGGTGCTGGACCTGAGTGAGAACTTCCTGTATGACTGCATCACCAAAACCAAAG
CCTTCTACGGCCTGGCCCGGCTGCGCAGACTCAACCTGTCCTTCAATTATCATAAGAAGGTGTCCTTTGCC
CACCTGCATCTGGCATCCTCCTTCGGGAGCCTACTGTCCCTGCAGGAGCTGGACATACATGGCATCTTCTT
CCGCTCGCTCAGCGAGACCACGCTCCAGTCGCTGGCCCACCTGCCCATGCTCCAGCGTCTGCATCTGCAGT
TGAACTTTATCAGCCAGGCCCAGCTCAGCATCTTCGGCGCCTTCCCTGGCCTGCGGTACGTGGACTTGTCA
GACAACCGCATCAGTGGAGCTGCAGAGCCCGCGGCTGCCACAGGGGAGGTAGAGGCGGACTGTGGGGAGAG
AGTCTGGCCACAGTCCCGGGACCTTGCTCTGGGCACACTGGGCACCCCCGGCTCAGAGGCCTTCATGCCGA
GCTGCAGGACCCTCAACTTCACCTTGGACCTGTCTCGGAACAACCTAGTGACTGTTCAGCCAGAGATGTTT
GTCCGGCTGGCGCGCCTCCAGTGCCTGGGCCTGAGCCACAACAGCATCTCGCAGGCGGTCAATGGCTCGCA
GT TCGTGCCTCTGAGCAACCTGCGGGTGCTGGACCTGTCCCATAACAAGCTGGACCTGTACCACGGGCGCT
CGTTCACGGAGCTGCCGCGGCTGGAGGCCTTGGACCTCAGCTACAACAGCCAGCCCTTCAGCATGCGGGGC
GTGGGCCACAATCTCAGCTTTGTGGCACAGCTGCCAGCCCTGCGCTACCTCAGCCTGGCGCACAATGGCAT
CCACAGCCGCGTGTCCCAGCAGCTCCGCAGCGCCTCGCTCCGGGCCCTGGACTTCAGTGGCAATACCCTGA
GCCAGATGTGGGCCGAGGGAGACCTCTATCTCCGCTTCTTCCAAGGCCTGAGAAGCCTGGTTCAGCTGGAC
CTGTCCCAGAATCGCCTGCATACCCTCCTGCCACGCAACCTGGACAACCTCCCCAAGAGCCTGCGGCTCCT
GCGGCTCCGTGACAATTACCTGGCTTTCTTCAACTGGAGCAGCCTGGCCCTCCTACCCAAGCTGGAAGCCC
TGGACCTGGCGGGAAACCAGCTGAAGGCCCTGAGCAATGGCAGCTTGCCCAACGGCACCCAGCTCCAGAGG
CTGGACCTCAGCGGCAACAGCATCGGCTTCGTGGTCCCCGGCTTTTTTGCCCTGGCCGTGAGGCTTCGAGA
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GC TCAACC TCAGCGCCAACGCCCTCAAGACGGTGGAGCCCTCCTGGTT TGGTTCCCTGGCGGGTGCCCTGA
AAGTCCTAGACGTGACCGCCAACCCCTTGCATTGCGCTTGCGGCGCAACCTTCGTGGACTTCTTGCTGGAG
GTGCAGGCTGCGGTGCCCGGCCTGCCTAGCCGTGTCAAGTGCGGCAGCCCGGGCCAGCTCCAGGGCCGCAG
CATCTTCGCACAGGACCTGCGCCTCTGC TTCCTGGACCTGGGGCTCTATCTCTTTGCTGGGACTGCACCGG
CAGTGCTGCTGCTGCTGGTGGTGCCGGTGGTGTACCACCGCGCCTACTGGAGGCTGAAGTACCACTGGTAC
CTTCTGCGGTGCTGGGTCAACCAGCGGTGGCGGCGGGAGGAAAAGTGCTACCTCTATGACAGCTTTGTGTC
CTACAATTCAGCTGATGAAAGTTGGGTGTTGCAGAAGCTGGTGCCTGAGCTGGAGCACGGTGCCTTCCGCC
TCTGCTTGCACCACCGCGACTTCCAGCCGGGCCGCAGCATCATTGACAACATTGTGGATGCTGTCTACAAC
AGCCGGAAGACGGTGTGCGTGGTAAGCCGCAGCTACCTGCGCAGCGAGTGGTGCTCTCTAGAGGTGCAGTT
GGCCAGCTACCGGCTGTTGGATGAGCGGCGTGACATCCTGGTACTGGTGCTGCTGGAGGACGTGGGTGATG
CTGAGCTGTCTGCCTACCACCGCATGCGGCGGGTGCTGCTGCGGCGCACCTACCTGCGCTGGCCTCTTGAC
CCCGCAGCTCAGCCGCTCTTTTGGGCACGGCTGAAGAGGGCACTGAGGTGGGGAGAGGGAGGAGAGGAGGA
GGAAGAAGAAGGTTTGGGTGGAGGGACGGGAAGGCCCAGGGAAGGAGACAAACAGATGTAGGCGGCCGC
MGPCRGALHPL SLLVQAAALALALAQGTLPAFL PCELQPHGLVNCNWL FLK SVPRFSAAAPRGNVT S L SLY
SNRIHHLHDYDFVHFVHLRRLNLKWNCPPASLS PMHFPCHMT IEPNTFLAVPTLEDLNLSYNS I TTVPAL P
SSLVSLSL SRTNI LVLDPATLAGLYALRFL FLDGNCYYKNPCQQALQVAPGALLGLGNLTHL SLKYNNLTV
VPRGL PP SLEYLLL SYNHI I TLAPEDLANLTALRVLDVGGNCRRCDHARNPCRECPKGFPQLHPNTFGHL S
HLEGLVLRDSSLYSLDPRWFHGLGNLMVLDLSENFLYDC I TKTKAFYGLARLRRLNL S FNYHKKVSFAHLH
LAS SFGSL L SLQEL DIHGI FFRSL SET TLQ SLAHL PMLQRLHLQLNF I SQAQLS I
FGAFPGLRYVDL SDNR
I SGAAEPAAATGEVEADCGERVWPQSRDLALGTLGT PGSEAFMPSCRTLNFTLDLSRNNLVTVQPEMFVRL
ARLQCLGL SHNS I SQAVNGSQFVPLSNLRVLDL SHNKLDLYHGRSFTELPRLEALDLSYNSQPFSMRGVGH
NL SFVAQL PALRYL SLAHNGIHSRVSQQLRSASLRALDFSGNTLSQMWAEGDLYLRFFQGLRSLVQLDLSQ
NRLHTLLPRNLDNL PKSLRLLRLRDNYLAFFNWSSLALL PKLEALDLAGNQLKALSNGSLPNGTQLQRLDL
SGNS I GFVVPGFFALAVRLRELNL SANALKTVE P SWFGS LAGALKVLDVTANPLHCACGATFVDFLLEVQA
AVPGL PSRVKCGSPGQLQGRS I FAQDLRLCFLDLGLYLFAGTAPAVLLLLVVPVVYHRAYWRLKYHWYLLR
CWVNQRWRREEKCYLYDSFVS YNSADE SWVI,QKLVPELEHGAFRI,CLHHRDFQPGRS I IDNIVDAVYNSRK
TVCVVSRSYLRSEWCSLEVQ_LASYRI,LDERRDILVLVI,LEDVGDAELSAYHRMRRVI,LRRTYLRWPI,DPAA
QPLFWARLKRALRWGEGGEEEEEEGLGGGTGRPREGDKQM
Example 6
Transfection of MDCK-pNilTyhyg-SEAP with canine TLR9-21 fusion constructs
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a) Transfection and selection of clones
To investigate the potential of MDCK-pNifTyhyg-SEAK-clonel5 for the expression
and
detection of TLR9-21 fusion constructs, this clonal cell line was transfected
with pIRES-puro-
canineTLR9-21. Transfectants were selected by repeated passage with medium
supplemented
5 with 300 ug/m1hygromycin and 8 ug/m1puromycin. Testing of the resulting
polyclonal cell line
with the standard oligonucleotide ODN-2006-PTO indicated induction of SEAP
secretion. Single
cell cloning was performed and 54 clones were expanded for further testing
with ODN-2006-
PTO (see graph below). A large number of clones were identified that showed
induction of
massive amounts of SEAP. Four clones with the best signal-to-noise ratio (No
17, 23, 32 and 40)
10 were chosen for expansion and to generate frozen stabilates. After
retesting, clone No 17 was
selected for further experiments (See figure 5).
b) MDCK-pNilTyhyg-SEAP-pIRESpuro-canTLR9-21-clone 17: testing of the
stimulatory
activity of oligonucleotides
Experiment 1:
A series of phosphorothioate oligonucleotides (PTO-ODNs, thio5-4 up to thio 5-
10) was tested,
together with the standard oligonucleotides from human medicine, 2006-PTO and
2007-PTO
EC50 (nM) Vmax
(mOD/min
/20111)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.3 311
2007¨PTO tcgtcgttgtcgttttgtcgtt 8.6 325
thio5-4 gtcgtcgtcgtc >250 n.
d.
thio5-5 gtcgtcgtcgtcgtc 80.6 348
thio5-6 gtcgtcgtcgtcgtcgtc 23.6 319
thio5-7 gtcgtcgtcgtcgtcgtcgtc 7.2 300
thio5-8 gtcgtcgtcgtcgtcgtcgtcgtc 7.8 308
thio5-10 gtcgtcgtcgtcgtcgtcgtcgtcgtcgtc 4.3 296
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From this experiments, it can be deduced that the pIRESpuro-canTLR9-21-
expressing
MDCK-pNifTyhyg cell line that was generated is capable of defining different
potencies via the
calculation of EC50 values (see figure 6).
It can be shown that for immunostimulatory ODNs, with structure elements such
as
gtcgtc, the number of cg elements is important: 9>7>6>5>>4>>3 (see table
above). This
experiment also immediately outlines the potential of this cell line for
generation of structure-
activity relationships (SAR) and lead optimization of immunostimulatory ODNs.
It was also shown that PTO-ODNs (2006-PTO and 2007-PTO) known from human
research are highly potent on the canine TLR9-21 fusion protein, but at least
one candidate
(thio5-10) of the inventor's initial 'lead optimization' proved to be as
potent or slightly more
potent on canine TLR9-21 fusion that these human standard ODNs.
Experiment 2:
A series of phosphodiester oligonucleotides (10 PDE-ODNs), that had proven to
be highly potent
on chicken TLR21 (the donor of the fusion part) were tested on canine TLR9-21
fusion, together
with the standard oligonucleotide from human medicine, 2006-PTO (see figure
7).
Result: None of the 10 PDE-ODNs showed any SEAP-inducing activity in the test
range (500
nM ¨ 3.9 nM), while 2006-PTO showed an EC50 of ¨ 4 nM, the expected range for
this ODN.
Interpretation: the recognition of ODNs is dictated by the N-terminal fusion
portion (in this case
canine TLR9). There is a dramatic species specificity of PDE-ODN recognition,
since the tested
ODNs show single digit nM or even pM EC50 values on chicken TLR21, while 2006-
PTO is less
potent on this receptor (31 nM) than on canine TLR9-21 fusion.
Experiment 3:
Here an experiment was performed on PTO-ODNs that have been used in the
literature in the
human and mouse context: 1668-PTO, 2216-PTO and 2395-PTO.
EC50 (nM) Vmax
(mOD/mi
n/20u1)
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1668¨PTO tccatgacgttcctgatgct 22.5 363
2216¨PTO gggggacgatcgtcgggggg 30.6 161
2395¨PTO tcgtcgttttcggcgcgcgccg 47.1 201
All three PTO-ODNs are active on canine TLR9-21 fusion with double digit nM
EC50 values.
However, with respect to maximally attainable stimulation of SEAP production
(Vmax) the
potency order is 1668 > 2395 >2216 (see figure 8). Remarkable, testing of the
same ODNs on
chicken TLR21 shows that 2216-PTO is inactive, 1668-PTO has an EC50 of ¨ 1000
nM, and only
2395-PTO has some potency in showing an EC50 of 39.4 nM.
Experiment 4:
Alead optimization' was attempted based on what could possibly be active
elements form the
published oligonucleotides 1668-PTO, 2216-PTO and 2395-PTO. These active
elements are
underlined and/or in Italic in the parental ODN and then arranged in repeats
in the mod1/2
ODNs:
EC50 (nM) Vmax
(mOD/mi
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.3 311
1668¨PTO tccatgacgttcctgatgct 22.5 363
1668¨modl gacgttgacgttgacgttgacgtt 5.7 312
1668¨mod2 tgacgttctgacgttctgacgttctgacgttc 4.9 273
2216¨PTO gggggacgatcgtcgggggg 30.6 161
2216¨modl gacgatcgtcgacgatcgtc 19.0 309
2216¨mod2 gacgatcgtcgacgatcgtcgacgatcgtc 7.9 293
2395¨PTO tcgtcgttttcggcgcgcgccg 47.1 201
2395¨modl tcgtcgttttcgtcgtcgttttcg 7.1 299
2395¨mod2 tcgtcgttttcgtcgtcgttttcgtcgtcgttttcg 3.7 258
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Based on the EC50 values the 'lead optimization' has proven to be successful.
In all three cases,
the mod1/2 PTO-ODN versions were more potent than their parental PTO ODN. In
the case of
2216-PTO and 2395-PTO in addition a significant increase in Vmax was visible.
Five out of six
newly designed PTO-ODNs are within the activity region of the paradigmal 2006-
PTO (see
figure 9).
Experiment 5:
A 'lead optimization' based on possible active elements was tried for the
published
oligonucleotide 2007-PTO.
EC50 (nM) Vmax
(mOD/mi
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.3 311
2007¨PTO tcgtcgttgtcgttttgtcgtt 8.6 325
2007¨modl tcgtcgttgtcgttttgtcgttgtcgtt 6.0 299
2007¨mod2 tcgtcgtcgtcgttgtcgttttgtcgtt 6.1 306
2007¨mod3 tcgtcgtcgtcgttgtcgttttgtcgttgtcgtt 6.6 295
2007¨dimer
tcgtcgttgtcgttttgtcgtttcgtcgttgtcgttttgtcgtt 4.1 293
The addition of cg-containing elements to the 5'-end or 3'-end or both, as
well as dimerization of
2007-PTO did not lead to an improvement of activity (both with respect to EC50
and V.), but
neither did it lead to a loss of activity. The single digit nanomolar activity
is preserved. None of
the listed ODNs has been reported in the literature so far (see figure 10).
Experiment 6:
Here two PTO-ODNs were tested that are mentioned in the literature (ODN-17 and
ODN-Lingl).
Also was tested the impact of replacement of the complete (thio5-8pde2A) and
partial (thio5-
8pde2B) phosphorothioate bonds of the CpG elements in thio5-8. Furthermore,
multimers of the
immunomodulatoy element ¨ttcgtc- were tested.
EC50 (nM) Vmax
(mOD/mi
n/20u1)
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2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.3 311
thio5-8 gtcgtcgtcgtcgtcgtcgtcgtc 7.0 276
thio5-8pde2A gtCGtCGtCGtCGtCGtCGtCGtc >100 n.
d.
thio5-8pde2B gtCGtcgtCGtcgtCGtcgtCGtc 61.0 268
thio9-3 ttcgtcttcgtcttcgtc 47.0 327
thio9-5 ttcgtcttcgtcttcgtcttcgtcttcgtc 8.9 248
ODN-17 gtcgttgtcgttgtcgtt 43.4 374
ODN¨Lingl tcgacgtttgacgtttgacgtt 8.2 359
It has been reported in the literature that replacement of PTO by PDE bonds
within the CpG
elements sometimes boosts stimulatory activity of PTO-ODNs. This idea was
tested with thio5-8.
In the inventor's hands the PDE-modified versions were far less active on
canine TLR9-21 fusion
than the parental 'PTO-only' ODN. They have identified one further novel PTO-
ODN being
similarly active on canine TLR9-21 fusion as 2006-PTO: thio9-5. Furthermore
Lingl-PTO
proved to be a potent PTO-ODN (see figure 11).
Experiment 7:
Here some PTO versions of PDE oligonucleotides were tested, that proved to be
highly active on
chicken TLR21, when combined with 5'- and 3'dG runs. The PTO versions,
however, lack the
5'- and 3'dG runs (therefore minus G, 'mG')
EC50 (nM) Vmax
(mOD /mi
n/20111)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.3
311
X4qu¨PT0mG ttcgttttcgttttcgttttcgtt 12.2
328
X4pe¨PT0mG ttcgttttcgttttcgttttcgttttcgtt 5.3
313
X4¨I¨tr¨PT0mG tttcgttttttcgttttttcgttt 10.1
332
X4¨I¨qu¨PT0mG tttcgttttttcgttttttcgttttttcgttt 6.2 262
X4¨II¨tr¨PT0mG ttttcgttttttttcgttttttttcgtttt 11.2
299
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All tested ODNs proved to be highly potent with EC50 and Vmax values identical
or close to the
standard PTO-ODN 2006 (see figure 12).
5 Experiment 8:
Further series of PTO-ODNs based on the active elements (chicken TLR21) of
ODNs X4, X43,
Z11 and CC-X were tested for their potency on canine TLR9-21 fusion
EC50 (nM) Vmax
(mOD /mi
10
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 4.0 292
X4-24¨PTO ttcgttttcgttttcgttttcgtt 5.4 274
X43-24¨PTO ttcgtcttcgtcttcgtcttcgtc 7.8 233
Z11-24¨PTO ctcgtcctcgtcctcgtcctcgtc 9.0 237
15 CC¨X-24¨PTO ttcgccttcgccttcgccttcgcc 10.5
235
X4-30¨PTO ttcgttttcgttttcgttttcgttttcgtt 3.6 280
X43-30¨PTO ttcgtcttcgtcttcgtcttcgtcttcgtc 3.6 231
Z11-30¨PTO ctcgtcctcgtcctcgtcctcgtcctcgtc 1.9 238
CC¨X-30¨PTO ttcgccttcgccttcgccttcgccttcgcc 1.9 242
20 ODN¨Lingl tcgacgtttgacgtttgacgtt 5.3
295
All new PTO-oligonucleotides have high stimulatory activity (EC50 in the
single digit nanomolar
range) and comparable Vmax on canine TLR9-21, in the case of Z11-30-PTO and CC-
X-30-PTO
with EC50s below 2 nM. None of these PTO-ODNs has been mentioned yet in
relation to their
25 use in dogs (see figures 13 and 14).
Experiment 9:
In this experiment repeats of the frequently used immunostimulatory elements
gacgtt and gtcgtt
were tested for their potency on canine TLR9-21 fusion.
30 EC50
(nM) Vmax
(mOD /mi
n/20u1)
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2006-PTO tcgtcgttttgtcgttttgtcgtt
7.1 431
GACGTT-18-PTO gacgttgacgttgacgtt 16.1
450
GACGTT-24-PTO gacgttgacgttgacgttgacgtt 6.8
456
GACGTT-30-PTO gacgttgacgttgacgttgacgttgacgtt 1.9 419
GTCGTT-18-PTO gtcgttgtcgttgtcgtt 12.4 397
GTCGTT-24-PTO gtcgttgtcgttgtcgttgtcgtt 5.9
369
GTCGTT-30-PTO gtcgttgtcgttgtcgttgtcgttgtcgtt 9.3 416
2216-PTO gggggacgatcgtcgggggg 1.2 187
1 0 In the case of the repeat element gacgtt, the experiment demonstrates
that repeat number matters.
The pentamer reaches an EC50 below 2 nM. Somewhat unexplained is the low EC50
of this new
batch of 2216-PTO. However, the Vmax value is clearly lower than for all other
ODNs tested (see
figure 15).
Experiment 10:
In this experiment repeats of triplet and quadruplet elements were tested for
their potency on
canine TLR9-21 fusion.
EC50 ( nM) Vmax
(mOD/min
/20u1)
2006-PTO tcgtcgttttgtcgttttgtcgtt
4.0 292
Thio-ACG-8 acgacgacgacgacgacgacgacg
inactive
Thio-TCG-8 tcgtcgtcgtcgtcgtcgtcgtcg
2.1 246
Thio-TCGT-6 tcgttcgttcgttcgttcgttcgt
7.2 256
25 Thio-TCGC-6 tcgctcgctcgctcgctcgctcgc 3.0 238
Thio-TCGA-6 tcgatcgatcgatcgatcgatcga
12.4 302
Thio-TCGG-6 tcggtcggtcggtcggtcggtcgg
4.0 226
Thio-CCGT-6 ccgtccgtccgtccgtccgtccgt
2.2 131
Thio-ACGT-6 acgtacgtacgtacgtacgtacgt
5.9 266
30 Thio-GCGT-6 gcgtgcgtgcgtgcgtgcgtgcgt 4.9 194
Thio-ACGA-6 acgaacgaacgaacgaacgaacga
5.1 282
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Thio¨CCGC-6 ccgcccgcccgcccgcccgcccgc poorly active*
Thio¨GCGC-6 gcgcgcgcgcgcgcgcgcgcgcgc poorly active*
Thio¨GCGG-6 gcgggcgggcgggcgggcgggcgg poorly active*
* EC50 and V. calculations not possible due to poor activity
It is remarkable that while TCG-8 is highly active, ACG-8 does not show any
stimulation
of canine TLR9-21 fusion. In this study, the `SAR' of PTO-ODN TCGT-6 was
investigated in
detail; the 5'T and then the 3'T was replaced with all other bases.
Unexpectedly, all the
derivatives proved to be highly active with respect to EC50, no dramatic loss
of activity could be
seen. With respect to V., a major loss of potency was seen for CCGT-6, a minor
one for
GCGT-6. The G/C-only containing PTO-ODNs were only marginally active in this
assay.
This is the determination of a comprehensive structure-activity relationship
for
canTLR9-21 based on hexamers of tetranucleotide motifs (see figures 16 and
17).
Experiment 11:
In this experiment combinations of immunomodulatory hexamer and tetramer
sequences
elements were tested for their potency on canine TLR9-21 fusion (see figure
18).
EC50 ( nM) Vmax
(mOD/min
/20u1)
P101 gtcgtcgtcgtcgtcgtcgtcgtc 38.2 226
P102 gtcgttgtcgttgtcgttgtcgtt 27.8 253
P103 gacgttgacgttgacgttgacgtt 23.6 265
P107 gtcgttgtcgacgtcgttgtcgac 29.4 299
P108 gacgttgtcgttgacgttgtcgtt 21.7 319
P109 tcgtgtcgtttcgtgtcgtttcgt 47.2 286
P1010 tcgtgacgtttcgtgacgtttcgt 41.0 335
P1011 gtcgtttcgtgtcgtttcgtgtcgtt 33.9 201
P1012 gacgtttcgtgacgtttcgtgacgtt 32.2 177
PTP13 gtcgttgtcgtcgtcgttgtcgtc 49.7 230
P1014 gacgttgtcgtcgacgttgtcgtc 30.2 231
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Conclusion: it is shown that MDCK-pNifTyhyg-SEAP-pIRESpuro-canTLR9-21 is a
unique
screening tool for the identification of canine TLR9 ligands. A number of
novel active ODNs has
been identified.
Example 7
Transfection of MDCK-pNilTyhyg-SEAP with porcine TLR9-21 fusion constructs
a) Transfection and selection of clones
MDCK-pNifTyhyg-SEAK-clonel5 was transfected with pIRES-puro-porcineTLR9-21.
Transfectants were selected by repeated passage with medium supplemented with
300 ug/m1
hygromycin and 8 ug/m1puromycin. Testing of the resulting polyclonal cell line
with the
standard oligonucleotide ODN-2006-PTO indicated induction of SEAP secretion.
Single cell
cloning was performed and 75 clones were expanded for further testing with ODN-
2006-PTO. A
large number of clones were identified that showed induction of SEAP. A number
of clones with
the best signal-to-noise ratio were chosen for expansion and to generate
frozen stabilates. After
retesting, clone No 20 was selected for further experiments (see figure 19).
b) MDCK-pNilTyhyg-SEAP-pIRESpuro-porcine TLR9-21-clone 20: testing of the
stimulatory activity of oligonucleotides
Experiment 1:
A series of phosphorothioate oligonucleotides (PTO-ODNs, thio5-4 up to thio 5-
10, thio9-3 and
thio9-5) was tested, together with the standard oligonucleotides from human
medicine, 2006-
PTO
EC50 (nM) Vmax
(mOD/mi
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.1 365
thio5-4 gtcgtcgtcgtc 673 458
thio5-5 gtcgtcgtcgtcgtc 436 489
thio5-6 gtcgtcgtcgtcgtcgtc 175 459
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thio5-7 gtcgtcgtcgtcgtcgtcgtc 127 470
thio5-8 gtcgtcgtcgtcgtcgtcgtcgtc 43.2 426
thio5-10 gtcgtcgtcgtcgtcgtcgtcgtcgtcgtc 45.4 388
thio5-8(II) gtcgtcgtcgtcgtcgtcgtcgtc 54.9 440
5 thio5-8pde2A gtCGtCGtCGtCGtCGtCGtCGtc 1810 553
thio5-8pde2B gtCGtcgtCGtcgtCGtcgtCGtc 159 343
thio9-3 ttcgtcttcgtcttcgtc 217 214
thio9-5 ttcgtcttcgtcttcgtcttcgtcttcgtc 73.9 390
From this experiments, it can be deduced that the pIRESpuro-porcineTLR9-21-
expressing MDCK-pNifTyhyg cell line that was generated is capable of defining
different
potencies via the calculation of EC50 values.
It is shown that for immunostimulatory ODNs, with structure elements such as
gtcgtc,
the number of cg elements is important: 9-7>6>5>4>3 (see table above). This
experiment also
immediately outlines the potential of this cell line for generation of
structure-activity
relationships (SAR) and lead optimization of immunostimulatory ODNs.
It was also shows that 2006-PTO known from human research are highly potent on
the
porcine TLR9-21 fusion protein. Furthermore, the impact of replacement of the
complete (thio5-
8pde2A) and partial (thio5-8pde2B) phosphorothioate bonds of the CpG elements
in thio5-8 was
tested. In the inventor's hands the PDE-modified versions were far less active
on porcine TLR9-
21 fusion than the parental 'PTO-only' ODN. Finally, thio9-3 and thio9-5,
which are trimers and
tetramers of the motif ttcgtc, respectively were tested. (Results, see figure
20).
Experiment 2:
Here some PTO versions of PDE oligonucleotides were tested that proved to be
highly active on
chicken TLR21, when combined with 5'- and 3'dG runs. The PTO versions,
however, lack the
5'- and 3'dG runs (therefore minus G, 'mG')
EC50 (nM) Vmax
(mOD /mi
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.1
365
X4qu¨PT0mG ttcgttttcgttttcgttttcgtt 42.1
434
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X4pe-PT0mG
ttcgttttcgttttcgttttcgttttcgtt 34.3 413
X4-I-tr-PT0mG tttcgttttttcgttttttcgttt 46.9
419
X4-I-qu-PT0mG tttcgttttttcgttttttcgttttttcgttt 35.8 417
X4-II-tr-PT0mG ttttcgttttttttcgttttttttcgtttt 35.5
345
5 ODN-17-PTO gtcgttgtcgttgtcgtt 119
459
ODN-Lingl-PTO tcgacgtttgacgtttgacgtt 8.1
391
X4-pent-PDE
GGGGGGTTCGTTTTCGTTTTCGTTTTCGTTTTCGTTGGGGG >500
10 Furthermore, two PTO-ODNs from published reports (ODN17 and ODN-Lingl)
were tested.
While the X4- X4-I- and X4-II- PTO-derivatives all proved to be potently
active with EC50
values between 30 and 50 nM, X4-pent-PDE, which is highly potent on chicken
TLR21 proved
to be a poor stimulator, both with respect to EC50 and prospective Vmax.
Interestingly, while
ODN-17-PTO ( a gtcgtt triplett) had an EC50 above 100 nM, ODN-Lingl -PTO
proved to be as
15 active as 2006-PTO (see figure 21).
Experiment 3:
In this experiment repeats of triplet and quadruplet elements were tested for
their potency on
porcine TLR9-21 fusion.
20 EC50
(nM) Vmax
(mop/min
/20111)
2006-PTO tcgtcgttttgtcgttttgtcgtt
6.1 365
Thio-ACG-8 acgacgacgacgacgacgacgacg
poorly active*
25 Thio-TCG-8 tcgtcgtcgtcgtcgtcgtcgtcg 61.8 246
Thio-TCGT-6 tcgttcgttcgttcgttcgttcgt
3.7 256
Thio-TCGC-6 tcgctcgctcgctcgctcgctcgc
50.9 238
Thio-TCGA-6 tcgatcgatcgatcgatcgatcga
104.9 302
Thio-TCGG-6 tcggtcggtcggtcggtcggtcgg
56.0 226
30 Thio-CCGT-6 ccgtccgtccgtccgtccgtccgt 568.0 131
Thio-ACGT-6 acgtacgtacgtacgtacgtacgt
163.2 266
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Thio¨GCGT-6 gcgtgcgtgcgtgcgtgcgtgcgt 85.1 194
Thio¨ACGA-6 acgaacgaacgaacgaacgaacga 72.1 282
Thio¨CCGC-6 ccgcccgcccgcccgcccgcccgc poorly active*
Thio¨GCGC-6 gcgcgcgcgcgcgcgcgcgcgcgc poorly active*
Thio¨GCGG-6 gcgggcgggcgggcgggcgggcgg poorly active*
* EC50 and 1.4õ,õ calculations not possible due to poor activity
ACG-8 does not show only minor stimulation of porcine TLR9-21 fusion, while
TCG-8 has an
EC50 of 62 nM. In this study, the `SAR' of PTO-ODN TCGT-6 was investigated in
detail; the
5'T and then the 3'T was replaced with all other bases. It turned out that
with respect to EC50,
TCGT-6 was clearly the best derivative, performing even better than the
standard 2006-PTO.
Like for canTLR9-21, with respect to V, a major loss of potency on porcineTLR9-
21 was seen
for CCGT-6, a minor one for GCGT-6. The G/C-only containing PTO-ODNs were only
marginally active in this assay, with GCGC-6 being the best one.
This is the determination of a comprehensive structure-activity relationship
for
porcineTLR9-21 based on hexamers of tetranucleotide motifs (see figures 22 and
23).
Experiment 4:
Here a lead optimization was attempted based upon possibly active elements
form the published
oligonucleotides 1668-PTO, 2216-PTO and 2395-PTO. These 'active elements' are
underlined
and/or in Italic in the parental ODN and then arranged in repeats in the
mod1/2 ODNs:
EC50 (nM) Vmax
(mOD /mi
n/20u1)
2006¨PTO tcgtcgttttgtcgttttgtcgtt 6.7 410
2007¨PTO tcgtcgttttgtcgttttgtcgtt 27.3 376
1668¨PTO tccatgacgttcctgatgct 17.0 388
1668¨modl gacgttgacgttgacgttgacgtt 10.0 457
1668¨mod2 tgacgttctgacgttctgacgttctgacgttc 16.5 434
2216¨PTO gggggacgatcgtcgggggg 249.0 361
2216¨modl gacgatcgtcgacgatcgtc 53.1 447
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2216-mod2 gacgatcgtcgacgatcgtcgacgatcgtc 19.0 448
2395-PTO tcgtcgttttcggcgcgcgccg 16.7 301
2395-modl tcgtcgttttcgtcgtcgttttcg 15.1 391
2395-mod2
tcgtcgttttcgtcgtcgttttcgtcgtcgttttcg 12.3 407
Results: see figures 24, 25 and 26.
Experiment 5:
In this experiment repeats of the frequently used immunostimulatory elements
gacgtt and gtcgtt
were tested for their potency on porcine TLR9-21 fusion.
EC50 (nM) Vmax
(mOD/mi
n/20u1)
2006-PTO tcgtcgttttgtcgttttgtcgtt 6.7 410
GACGTT-18-PTO gacgttgacgttgacgtt 60.2
405
GACGTT-24-PTO gacgttgacgttgacgttgacgtt 34.0
421
GACGTT-30-PTO gacgttgacgttgacgttgacgttgacgtt 30.2 399
GTCGTT-18-PTO gtcgttgtcgttgtcgtt 34.6
325
GTCGTT-24-PTO gtcgttgtcgttgtcgttgtcgtt 20.0 354
GTCGTT-30-PTO gtcgttgtcgttgtcgttgtcgttgtcgtt 21.6 347
X4-pent-PDE
GGGGGGTTCGTTTTCGTTTTCGTTTTCGTTTTCGTTGGGGG >500 ----
Results: see figure 27.
Experiment 6:
Here a 'lead optimization' was attempted based upon possible active elements
form the published
oligonucleotide 2007-PTO.
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EC50 (nM) Vmax
(mOD/min
/20u1)
2006-PTO tcgtcgttttgtcgttttgtcgtt 6.7 410
2007-PTO tcgtcgttgtcgttttgtcgtt 27.3 378
2007-modl tcgtcgttgtcgttttgtcgttgtcgtt 32.7 439
2007-mod2 tcgtcgtcgtcgttgtcgttttgtcgtt 28.5 436
2007-mod3 tcgtcgtcgtcgttgtcgttttgtcgttgtcgtt 31.7 425
2007-dimer
tcgtcgttgtcgttttgtcgtttcgtcgttgtcgttttgtcgtt 8.9 351
Results: see figure 28.
Experiment 7:
Further series of PTO-ODNs based on the active elements (chicken TLR21) of
ODNs X4, X43,
Z11 and CC-X were tested for their potency on porcine TLR9-21 fusion
EC50 (nM) Vmax
(mOD/mi
n/20u1)
2006-PTO tcgtcgttttgtcgttttgtcgtt 5.1 369
X4-24-PTO ttcgttttcgttttcgttttcgtt 22.5 479
X43-24-PTO ttcgtcttcgtcttcgtcttcgtc 36.9 454
Z11-24-PTO ctcgtcctcgtcctcgtcctcgtc 20.8 498
CC-X-24-PTO ttcgccttcgccttcgccttcgcc 92.2 469
X4-30-PTO
ttcgttttcgttttcgttttcgttttcgtt 24.4 447
X43-30-PTO
ttcgtcttcgtcttcgtcttcgtcttcgtc 23.9 383
Z11-30-PTO
ctcgtcctcgtcctcgtcctcgtcctcgtc 12.6 385
CC-X-30-PTO ttcgccttcgccttcgccttcgccttcgcc 47.3 395
Most new PTO-oligonucleotides have high stimulatory activity (EC50 in the
single digit
nanomolar range) and comparable Vmax on porcine TLR9-21. Particularly potent
appears to be the
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motif of Z11 (ctcgtc). None of these PTO-ODNs has been mentioned yet in the
context porcine
(see figures 29 and 30).
Experiment 8:
In this experiment combinations of immunomodulatory hexamer and tetramer
sequences
elements were tested for their potency on porcine TLR9-21 fusion.
EC50 (nM) Vmax
(mop/min
/20111)
P101 gtcgtcgtcgtcgtcgtcgtcgtc 55.9 499
P102 gtcgttgtcgttgtcgttgtcgtt 28.0 472
P103 gacgttgacgttgacgttgacgtt 49.1 503
P107 gtcgttgtcgacgtcgttgtcgac 23.7 460
P108 gacgttgtcgttgacgttgtcgtt 38.5 442
P109 tcgtgtcgtttcgtgtcgtttcgt 35.6 460
P1010 tcgtgacgtttcgtgacgtttcgt 22.4 427
P1011 gtcgtttcgtgtcgtttcgtgtcgtt 38.8 438
P1012 gacgtttcgtgacgtttcgtgacgtt 63.8 469
PTP13 gtcgttgtcgtcgtcgttgtcgtc 45.9 456
P1014 gacgttgtcgtcgacgttgtcgtc 42.4 479
In this experiment, various combinations of gtcgtc-, gtcgtt-, gtcgac- and tcgt-
containing PTO-
ODNs proved to be of similar double digit nanomolar potency on porcine TLR9-21
(see figure
31).
Example 8
Experimental design
Two groups of five (N=5) Beagle puppies of 3-4 months of age, not vaccinated
against Rabies, of
which the bitches were not vaccinated the past 12 month against Rabies were
used. The puppies
were vaccinated with 1 ml of the respective vaccine compositions. Immediately
before
vaccination (T=0) and at T-2, T-4, T-6, T-8, T-12, T=16, T=20 and T=24
weeks post
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virus were
determined.
Groups and vaccination
Dose Immunostimulator
Group N= Vaccine
volume Supplement Concentration
1 5 Nobivac0 Rabies 1 ml -
2 5 Nobivac0 Rabies 1 ml Thio-tcg-8-PTO 5 lag / 1
dose
5
Vaccine
Nobivae Rabies
Formulation : commercially available vaccine
Presentation : 10 ml flacons
1 0 Supplier : Intervet International BV, Boxmeer, The
Netherlands
Dosage and administration
The puppies were vaccinated subcutaneously (s.c.) in the neck with 1 ml of
Nobivac0 Rabies
vaccine with (group 2) or without (group 1) the addition of 5 lag Thio-tcg-8-
PTO
15 (TCGTCGTCGTCGTCGTCGTCGTCG) per 1 ml.
Induction of antibodies
Directly before vaccination (T=0) and at T-2, T-4, T-6, T-8, T-12, T=16,
T=20 and T=24
weeks post vaccination, blood samples were taken from each puppy. Blood
samples were
20 allowed to clot over night at 2-8 C. After centrifugation the serum was
transferred into
appropriate containers and stored at -20 C until analysis. The antibody titer
against Rabies in the
serum was determined using the Rapid Fluorescent Focus Inhibition Test (RFFIT)
which is a
virus neutralization test.
25 Rapid Fluorescent Focus Inhibition Test (RFFIT)
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RFFIT has been internationally recognized as the standard in vitro test for
quantifying the
presence of Rabies neutralizing antibodies. Serial 3-fold dilutions of the
sera to be examined
were made and mixed with an equal volume of a Rabies virus¨suspension
containing a standard
dose (according to WHO/PHEUR with a titer between 30-300 Focus Forming Units
(FFU)). The
sera/virus mixture were incubated at 37 C and 5% CO2 for 90 minutes. To grow
non-neutralized
virus after a pre-incubation period, susceptible cells (BHK cells) were added
into the mixture and
incubated for 24 hour at 37 C and 5% CO2 to form a monolayer. After incubation
and Rabies
virus specific immuno-staining, the monolayers were observed for fluorescent
foci by
microscopy after which the titers (in IU/ml) were calculated.
Results: As can be seen from figure 32, the anti-rabies virus titre found in
dogs that received the
Nobivac rabies vaccine and the CpG ODN Thio-teg-8-PTO
(TCGTCGTCGTCGTCGTCGTCGTCG) is three times the amount of that of the same
rabies
vaccine without this CpG ODN.
